Covalent microparticle-drug conjugates for biological targeting

ABSTRACT

This invention provides reagents and methods for specifically delivering antibiotic, antimicrobial and antiviral compounds, drugs and agents to phagocytic mammalian cells. The invention also relates to specific delivery to and uptake of such compounds by phagocytic cells. The invention specifically relates to reagents and methods for facilitating the entry of antibiotic, antimicrobial and antiviral compounds, drugs and agents into phagocytic cells. The invention specifically provides compositions of matter and pharmaceutical embodiments of such compositions comprising such antibiotic, antimicrobial or antiviral compounds, drugs and agents conjugated to, impregnated with or coated onto particulate carriers generally termed microparticles. In particular embodiments, the antibiotic, antimicrobial and antiviral compounds, drugs and agents are covalently linked to a microparticle via a specifically-degradable linker molecule which is the target of a microorganism-specific protein having enzymatic activity. Also provided are porous microparticles impregnated with antibiotic, antimicrobial or antiviral compounds, drugs and agents wherein the surface or outside extent of the microparticle is covered with a degradable coating that is specifically degraded within an infected phagocytic mammalian cell. Also provided are nonporous microparticles coated with antibiotic, antimicrobial or antiviral compounds, drugs and agents and further coated wherein the surface or outside extent of the microparticle is covered with a degradable coating that is specifically degraded within an infected phagocytic mammalian cell. Thus, the invention provides cell targeting of drugs wherein the targeted drug is only released in cells infected with a particular microorganism. Methods of inhibiting, attenuating, arresting, combating and overcoming microbial infection of phagocytic mammalian cells in vivo and in vitro, especially cells infected with tuberculosis-causing and other Mycobacterium species microorganisms, are also provided.

[0001] This invention was made with government support under grant1-R01-CA 49416 by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to reagents and methods for facilitatingthe entry of biologically-active compounds into phagocytic cells. Theinvention specifically provides particulate carriers generally termedmicroparticles comprising antimicrobial compounds, both per se ascompositions of matter and as pharmaceutical compositions thereof.Alternative embodiments of said microparticle carriers are providedwherein one or a multiplicity of antimicrobial compounds are linked to amicroparticle via a specifically-cleaved linker moiety, or wherein aporous microparticle is impregnated with one or a multiplicity ofantimicrobial compounds, or wherein the microparticle is coated with oneor a multiplicity of antimicrobial compounds, wherein the impregnated orcoated microparticle is further coated with a specifically-degradablecoating material, wherein in their respective embodiments thespecifically-cleaved linker moiety and the specifically-degradablecoating material are the targets of a microorganism-specific proteinhaving an enzymatic activity not otherwise expressed in the phagocyticcell, or that is specifically expressed by the phagocytic cell only wheninfected with said microorganism. Thus, the invention provides celltargeting of drugs to phagocytic cells wherein the targeted drug is onlyreleased in phagocytic cells that infected with a particularmicroorganism. Methods of treating diseases having an intracellularmicrobial etiology are also provided, particularly for the treatment oftuberculosis and other Mycobacterium-caused diseases.

[0004] 2. Background of the Related Art

[0005] A major goal in the pharmacological arts has been the developmentof reagents and methods for facilitating specific delivery oftherapeutic compounds, drugs and other agents to the appropriate cellsand tissues that would benefit from such treatment, and the avoidance ofthe general physiological effects of systemic or otherwise inappropriatedelivery of such compounds, drugs or agents to other cells or tissues ofthe body. The most common example of the need for such specificity is inthe field of antibiotic therapy, in which the amount of a variety ofantibiotic, antimicrobial and antiviral compounds, drugs and agents thatcan be safely administered to a patient is limited by their cytotoxicand immunogenic effects.

[0006] It is also recognized in the medical arts that certain cells arethe sites of pharmacological action of certain compounds, drugs oragents or are involved in the biological response to certain stimuli. Inparticular, it is now recognized that certain cell types are reservoirsfor occult infection that evades normal immune surveillance and permitsthe persistence of a chronically infected disease state. Specificdelivery of diagnostic or therapeutic compounds, drugs or agents to suchcells is thus desirable to increase the specificity and effectiveness ofclinical diagnostic or therapeutic techniques.

[0007] A. Drug Targeting

[0008] It is desirable to increase the efficiency and specificity ofadministration of a therapeutic compound, drug or agent to the cells ofthe relevant tissues in a variety of pathological states. This isparticularly important as relates to antibiotic, antimicrobial andantiviral compounds, drugs or agents. These compounds, drugs or agentstypically have pleiotropic antibiotic, immunogenic, cytopathic andcytotoxic effects that damage or destroy uninfected cells as well asinfected cells. In addition, certain compounds, drugs or agents are“activated” or chemically modified by an enzymatic or chemical activityspecific for infected cells, in which activated form the compounds,drugs or agents are particularly toxic. Resistance to these types ofcompounds, drugs or agents can arise by attenuation, mutation orablation of the chemical or enzymatic activity in the infected cell.Thus, an efficient delivery system which would enable the delivery ofsuch compounds, drugs or agents, particularly said “activated” formsthereof, specifically to infected cells would increase the efficacy oftreatment, overcome drug resistance, reduce the associated “sideeffects” of such drug treatments, and also serve to reduce morbidity andmortality associated with clinical administration of such compounds,drugs or agents.

[0009] Numerous methods for enhancing the cytotoxic activity and thespecificity of antibiotic drug action have been proposed. One method,receptor targeting, involves linking the therapeutic agent to a ligandwhich has an affinity for a receptor expressed on the desired targetcell surface. Using this approach, antibiotic, antimicrobial andantiviral compounds, drugs and agents are intended to adhere to thetarget cell following formation of a ligand-receptor complex on the cellsurface. Entry into the cell could then follow as the result ofinternalization of ligand-receptor complexes. Following internalization,the antibiotic, antimicrobial and antiviral compounds, drugs and agentsmay then exert therapeutic effects directly on the cell.

[0010] The ligand-receptor approach is plagued by a number of biologicallimitations. Receptor-mediated uptake does not specifically targetinfected cells; all cells that happen to express the receptor take upthe drug. A further limitation of the receptor targeting approach liesin the fact that there are only a finite number of receptors on thesurface of target cells. It has been estimated that the maximum numberof receptors on a cell is approximately one million (Darnell et al.,1990, Molecular Cell Biology, 2d ed., W. H. Freeman: N.Y.). Thisestimate predicts that there may be a maximum one milliondrug-conjugated ligand-receptor complexes on any particular cell. Sincenot all of the ligand-receptor complexes may be internalized, and anygiven ligand-receptor system may express many-fold fewer receptors onany particular cell surface, the efficacy of intracellular drug deliveryusing this approach is uncertain. Other known intracellularligand-receptor complexes (such as the steroid hormone receptor) expressas few as ten thousand hormone molecules per cell, and thus are evenless suitable for mediating cell-specific targeting of antibiotic,antibiotic or antiviral compounds, drugs and agents. Id. Finally, oncethe bound drug entered a cell, it would not be expected to bedifferentially released in infected cells.

[0011] Other methods of delivering therapeutic agents at concentrationshigher than those achievable through the receptor targeting processinclude the use of lipid conjugates that have selective affinities forspecific biological membranes. These methods have met with littlesuccess (see, for example, Remy et al., 1962, J. Org. Chem. 27:2491-2500; Mukhergee & Heidelberger, 1962, Cancer Res. 22: 815-22;Brewster et al., 1985, J. Pharm. Sci. 77: 981-985).

[0012] Liposomes have been used to attempt cell targeting

[0013] U.S. Pat. No. 5,223,263, issued Jun. 29, 1993 to Hostetler et al.disclose conjugates between antiviral nucleoside analogues and polarlipids.

[0014] U.S. Pat. No. 5,484,809, issued Jan. 16, 1996 to Hostetler et al.disclose taxol and taxol derivatives conjugated to phospholipids.

[0015] U.S. Pat. No. 5,580,571, issued Dec. 3, 1996 to Hostetler et al.disclose nucleoside analogues conjugated to phospholipids.

[0016] U.S. Pat. No. 5,744,461, issued Apr. 28, 1998 to Hostetler et al.disclose nucleoside analogues conjugated to phosphonoacetic acid lipidderivatives.

[0017] U.S. Pat. No. 5,744,592, issued Apr. 28, 1998 to Hostetler et al.disclose nucleoside analogues conjugated to phospholipids.

[0018] U.S. Pat. No. 5,756,116, issued May 26, 1998 to Hostetler et al.disclose nucleoside analogues conjugated to phospholipids.

[0019] International Patent Application Publication Number WO89/02733,published April 1989 to Vical disclose conjugates between antiviralnucleoside analogues and polar lipids.

[0020] European Patent Application Publication Number 0350287A2 to Vicaldisclose conjugates between antiviral nucleoside analogues and polarlipids.

[0021] International Patent Application Publication Number WO93/00910 toVical disclose conjugates between antiviral nucleoside analogues andpolar lipids.

[0022] Rahman et al., 1982, Life Sci. 31: 2061-71 found that liposomeswhich contained galactolipid as part of the lipid appeared to have ahigher affinity for parenchymal cells than liposomes which lackedgalactolipid.

[0023] Gregoriadis, 1995, Trends in Biotechnology 13: 527-537 reviewsthe “progress and problems” associated with using liposomes for targeteddrug delivery.

[0024] Ledley, 1995, Human Gene Therapy 6: 1129-1144 reviews the use ofliposomes for gene therapy.

[0025] Mickisch, 1995, World J. Urology 13: 178-185 reviews the use ofliposomes for gene therapy of renal cell carcinoma.

[0026] Yang et al. 1997, J. Neurotrauma 14: 281-297 review the use ofcationic liposomes for gene therapy directed to the central nervoussystem.

[0027] Storm & Crommelin, 1997, Hybridoma 16: 119-125 review thepreliminary use of liposomes for targeting chemotherapeutic drugs totumor sites.

[0028] Manusama et al., 1998, Semin. Surg. Oncol. 14: 232-237 reportedon preclinical and clinical trials of liposome-encapsulated tumornecrosis factor for cancer treatments.

[0029] To date, however, efficient or specific drug delivery has notbeen predictably achieved using drug-encapsulated liposomes.

[0030] Drug delivery to specific sites or cells has been attempted as away to enhance drug effectiveness. In one example of this approach,prodrug activation has been attempted using antibodies to provide“time-released” drug delivery agents (Bignami et al., 1992, Cancer Res.52: 5759-5764). In this approach, a specific targeting antibodyconjugated with a prodrug-activating enzyme was used to activate asystemically-delivered prodrug only at the specific site recognized bythe antibody.

[0031] There remains a need for the development of cell-specific drugtargeting and delivery systems, particularly with antibiotic,antimicrobial and antiviral compounds, drugs and agents.

[0032] B. Phagocytic Cell-Specific Targeting

[0033] Cell-specific targeting is an important goal of antimicrobialtherapy, particularly in the event that a specific cell type is a targetof acute or chronic infection. Targeting a specific infected cell typewould be advantageous because it would allow administration ofantibiotic, antimicrobial or antiviral compounds, drugs or agents to ananimal suffering from infection with a microbial pathogen, without therisk of non-specific toxicity to uninfected cells that would exist withnontargeted administration of toxic compounds, and because it wouldpermit administration of dosages unattainable usingsystemically-administered, non-targeted embodiments of such antibiotic,antiviral and antimicrobial compounds, drugs and agents. This isparticularly true of “activated” compounds, drugs or agents, which areby definition particularly toxic forms of said compounds, drugs oragents and particularly efficient in their antibiotic, antimicrobial, orantiviral properties. An additional advantage of such targetedantimicrobial therapy would be improved pharmacokinetics that wouldresult from specific concentration of antibiotic, antimicrobial orantiviral compounds, drugs and agents to the infected cells that are thesites of infection.

[0034] Phagocytic cells such as monocytes and macrophages are known tobe specific targets for infection of certain pathogenic microorganisms.

[0035] Sturgill-Koszycki et al., 1994, Science 263: 678-681 disclosethat the basis for lack of acidification of phagosomes in M. avium andM. tuberculosis-infected macrophages is exclusion of the vesicularproton-ATPase.

[0036] Sierra-Honigman et al., 1993, J. Neuroimmunol. 45: 31-36 discloseBoma disease virus infection of monocytic cells in bone marrow.

[0037] Maciejewski et al., 1993, Virol. 195: 327-336 disclose humancytomegalovirus infection of mononucleated phagocytes in vitro.

[0038] Alvarez-Dominguez et al., 1993, Infect. Immun. 61: 3664-3672disclose the involvement of complement factor C1q in phagocytosis ofListeria monocytogenes by macrophages.

[0039] Kanno et al., 1993, J. Virol. 67: 2075-2082 disclose thatAleutian mink disease parvovirus replication depends on differentiationstate of the infected macrophage.

[0040] Embretson et al., 1993, Nature 362: 359-362 disclose covertinfection of macrophages by human immunodeficiency virus.

[0041] Meltzer & Gendelman, 1992, Curr. Top. Microbiol. Immunol. 181:239-263 disclose infection of mononuclear phagocytes with humanimmunodeficiency virus.

[0042] Kanno et al., 1992, J. Virol. 66: 5305-5312 disclose thatAleutian mink disease parvovirus infects peritoneal macrophages in mink.

[0043] Narayan et al., 1992, J. Rheumatol. 32: 25-32 disclose arthritisin animals caused by infection of macrophage precursors with lentivirus,and activation of quiescent lentivirus infection upon differentiation ofsuch precursor cells into terminally-differentiated macrophages.

[0044] Horwitz, 1992, Curr. Top. Microbiol. Immunol. 181: 265-282disclose Legionella pneumophila infections of alveolar macrophages asthe basis for Legionnaire's disease and Pontiac fever.

[0045] Sellon et al., 1992, J. Virol. 66: 5906-5913 disclose that equineinfectious anemia virus replicates in tissue macrophages in vivo.

[0046] Groisman et al., 1992, Proc. Natl. Acad. Sci. USA 89: 11939-11943disclose that S. typhimurium survives inside infected macrophages byresistance to antibacterial peptides.

[0047] Friedman et al., 1992, Infect. Immun. 60: 4578-4585 discloseBordetella pertussis infection of human macrophages.

[0048] Stellrecht-Broomhall, 1991, Viral Immunol. 4: 269-280 disclosethat lymphocytic choriomeningitis virus infection of macrophagespromotes severe anemia caused by macrophage phagocytosis of red bloodcells.

[0049] Frehel et al., 1991, Infect. Immun. 59: 2207-2214 discloseinfection of spleen and liver-specific inflammatory macrophages byMycobacterium avium, the existence of the microbe in encapsulatedphagosomes within the inflammatory macrophages and survival therein inphagolysosomes.

[0050] Bromberg et al., 1991, Infect. Immun. 59: 4715-4719 discloseintracellular infection of alveolar macrophages.

[0051] Mauel, 1990, J. Leukocyte Biol. 47: 187-193 disclose thatLeishmania spp. are intracellular parasites in macrophages.

[0052] Buchmeier and Heffron, 1990, Science 248: 730-732 disclose thatSalmonella typhimurium infection of macrophages induced bacterial stressproteins.

[0053] Panuska et al., 1990, J. Clin. Invest. 86: 113-119 discloseproductive infection of alveolar macrophages by respiratory syncytialvirus.

[0054] Cordier et al., 1990, Clin. Immunol. Immunopathol. 55: 355-367disclose infection of alveolar macrophages by visna-maedi virus inchronic interstitial lung disease in sheep.

[0055] Schlessinger and Horwitz, 1990, J. Clin. Invest. 85: 1304-1314disclose Mycobacterium leprae infection of macrophages.

[0056] Schmidt et al., 1990, Res. Virol. 141: 143-152 disclose infectionof primary cultures of liver Kupffer cells with human immunodeficiencyvirus.

[0057] Clarke et al., 1990, AIDS 4: 1133-1136 disclose humanimmunodeficiency virus infection of alveolar macrophages in lung.

[0058] Baroni et al., 1988, Am. J. Pathol. 133: 498-506 disclose humanimmunodeficiency virus infection of lymph nodes.

[0059] Payne et al, 1987, J. Exp. Med. 166: 1377-1389 discloseMycobacterium tuberculosis infection of macrophages.

[0060] Murray et al., 1987, J. Immunol. 138: 2290-2296 disclose thatliver Kupffer cells are the initial targets for L. donovani infection.

[0061] Koenig et al., 1986, Science 233: 1089-1093 disclose humanimmunodeficiency virus infection of macrophages in the central nervoussystem.

[0062] Gendelman et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7086-7090disclose infection of phagocytic cells with lentivirus.

[0063] Horwitz and Maxfield, 1984, J. Cell Biol. 99: 1936-1943 disclosethat L. pneumophila survives in infected phagocytic cells at least inpart by inhibiting reduction of intraphagosomic hydrogen ionconcentration (pH).

[0064] Shanley and Pesanti, 1983, Infect. Immunol. 41: 1352-1359disclose cytomegalovirus infection of macrophages in murine cells.

[0065] Horwitz, 1983, J. Exp. Med. 158: 2108-2126 disclose that L.pneumophila is an obligate intracellular parasite that is phagocytizedinto a phagosome wherein fusion with lysosome is inhibited.

[0066] Chang, 1979, Exp. Parasitol. 48: 175-189 disclose Leischmaniadonovani infection of macrophages.

[0067] Wyrick and Brownridge, 1978, Infect. Immunol. 19: 1054-1060disclose Chlamydia psittaci infection of macrophages.

[0068] Halstead et al., 1977, J. Exp. Med. 146: 201-217 disclosedinfection of phagocytic cells with dengue virus.

[0069] Nogueira and Cohn, 1976, J. Exp. Med. 143: 1402-1420 discloseTrypanosoma cruzi infection of macrophages.

[0070] Jones and Hirsch, 1972, J. Exp. Med. 136: 1173-1194 discloseToxoplasma gondii infection of macrophages.

[0071] Persistent infection of phagocytic cells has been reported in theprior art.

[0072] Embretson et al., 1993, Nature 362: 359-361 disclose covertinfection of macrophages with HIV and dissemination of infected cellsthroughout the immune system early in the course of disease.

[0073] Schnorr et al., 1993, J. Virol. 67: 4760-4768 disclose measlesvirus persistent infection in vitro in a human monocytic cell line.

[0074] Meltzer and Gendelman, 1992, Curr. Topics Microbiol. Immunol.181: 239-263 provide a review of HIV infection of tissue macrophages inbrain, liver, lung, skin, lymph nodes, and bone marrow, and involvementof macrophage infection in AIDS pathology.

[0075] Blight et al., 1992, Liver 12: 286-289 disclose persistentinfection of liver macrophages (Kuppfer cells) by hepatitis C virus.

[0076] McEntee et al., 1991, J. gen. Virol. 72: 317-324 disclosepersistent infection of macrophages by HIV resulting in destruction of Tlymphocytes by fusion with infected macrophages, and that themacrophages survive fusion to kill other T lymphocytes.

[0077] Kondo et al., 1991, J. gen. Virol. 72: 1401-1408 disclose latentinfection by herpes simplex virus 6 of monocytes activated bydifferentiation into macrophages.

[0078] King et al., 1990, J. Virol. 64: 5611-5616 disclose persistentinfection of macrophages with lymphocytic choriomeningitis virus.

[0079] Schmitt et al., 1990, Res. Virol. 141: 143-152 disclose a rolefor HIV infection of Kupffer cells as reservoirs for HIV infection.

[0080] Gendelman et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7086-7090disclose lentiviral (visna-maedi) infection of bone marrow precursors ofperipheral blood monocytes/macrophages that provide a reservoir oflatently-infected cells.

[0081] Halstead et al., 1977, J. Exp. Med. 146: 201-217 disclose thatmacrophages are targets of persistent infection with dengue virus.

[0082] Mauel et al., 1973, Nature New Biol. 244: 93-94 disclose thatlysis of infected macrophages with sodium dodecyl sulfate could releaselive microbes.

[0083] Attempts at cell-specific drug targeting have been reported inthe prior art.

[0084] Rubinstein et al., 1993, Pharm. Res. 10: 258-263 report colontargeting using calcium pectinate (CaPec)-conjugated drugs, based ondegradation of CaPec by colon specific (i.e., microflora-specific)enzymes and a hydrophobic drug incorporated into the insoluble CaPecmatrices.

[0085] Sintov et al., 1993, Biomaterials 14: 483-490 reportcolon-specific targeting using conjugation of drug to insolublesynthetic polymer using disaccharide cleaved by enzymes made byintestinal microflora, specifically, β-glycosidic linkages comprisingdextran.

[0086] Franssen et al., 1992, J. Med. Chem. 35: 1246-1259 report renalcell/kidney drug targeting using low molecular weight proteins (LMWP) ascarriers, using enzymatic/chemical hydrolysis of a spacer moleculelinking the drug and LMWP carrier.

[0087] Bai et al., 1992, J. Pharm. Sci. 81: 113-116 report intestinalcell targeting using a peptide carrier-drug system wherein the conjugateis cleaved by an intestine-specific enzyme, prolidase.

[0088] Gaspar et al., 1992, Ann. Trop. Med. Parasitol. 86: 41-49disclose primaquine-loaded polyisohexylcyanoacrylate nanoparticles usedto target Leschmania donovani infected macrophage-like cells in vitro.

[0089] Pardridge, 1992, NIDA Res. Monograph 120: 153-168 reportopioid-conjugated chimeric peptide carriers for targeting to brainacross the blood-brain barrier.

[0090] Bai and Amidon, 1992, Pharm. Res. 9: 969-978 report peptide-drugconjugates for oral delivery and intestinal mucosal targeting of drugs.

[0091] Ashbom et al., 1991, J. Infect. Dis. 163: 703-709 disclose theuse of CD4-conjugated Pseudomonas aeruginosa exotoxin A to killHIV-infected macrophages.

[0092] Larsen et al., 1991, Acta Pharm. Nord. 3: 41-44 reportenzyme-mediated release of drug from dextrin-drug conjugates bymicroflora-specific enzymes for colon targeting.

[0093] Faulk et al., 1991, Biochem. Int. 25: 815-822 reportadriamycin-transferrin conjugates for tumor cell growth inhibition invitro.

[0094] Zhang and McCormick, 1991, Proc. Natl. Acad. Sci. USA 88:10407-10410 report renal cell targeting using vitamin B6-drugconjugates.

[0095] Blum et al., 1982, Int. J. Pharm. 12: 135-146 report polystyrenemicrospheres for specific delivery of compounds to liver and lung.

[0096] Trouet et al., 1982, Proc. Natl. Acad. Sci. USA 79: 626-629report that daunorubicin-conjugated to proteins was cleaved by lysosomalhydrolases in vivo and in vitro.

[0097] Shen et al., 1981, Biochem. Biophys. Res. Commun. 102: 1048-1052report pH-labile N-cis-acontinyl spacer moieties.

[0098] Monoclonal antibodies have been used in the prior art for drugtargeting.

[0099] Serino et al, U.S. Pat. No. 4,793,986, issued Dec. 27, 1988,provides platinum anticancer drugs conjugated to polysaccharide(dextrin) carrier for conjugation to monoclonal antibodies for tumorcell targeting.

[0100] Bickel et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2618-2622discloses the use of a chimeric protein vector for targeting acrossblood-brain barrier using an anti-transferrin monoclonal antibody.

[0101] Rowlinson-Busza and Epenetos, 1992, Curr. Opin. Oncol. 4:1142-1148 provides antitumor immunotargeting using toxin-antibodyconjugates.

[0102] Blakey, 1992, Acta Oncol. 31: 91-97 provides a review ofantitumor antibody targeting of antineoplastic drugs.

[0103] Senter et al., 1991, in Imunobiology of Peptides and Proteins,Vol. VI, pp. 97-105 discloses monoclonal antibodies linked to alkalinephosphatase or penicillin-V amidase to activate prodrugs specifically atsite of antibody targeting, for therapeutic treatment of solid tumors.

[0104] Drug-carrier conjugates have been used in the prior art toprovide time-release drug delivery agents.

[0105] Couveur and Puisieux, 1993, Adv. Drug Deliv. Rev. 10: 141-162provide a review of microcapsule (vesicular), microsphere (dispersedmatrix) and microparticle (1-250 82 m)-based drug delivery systems,based on degradation of particle with drug release, to provide timerelease of drugs, oral delivery via transit through the intestinalmucosa and delivery to Kupffer cells of liver.

[0106] Duncan, 1992, Anticancer Drugs 3: 175-210 provide a review ofimproved pharmacokinetic profile of in vivo drug release of anticancerdrugs using drug-polymer conjugates.

[0107] Heinrich et al., 1991, J. Pharm. Pharmacol. 43: 762-765 disclosepoly-lactide-glycolide polymers for slow release of gonadotropinreleasing hormone agonists as injectable implants.

[0108] Wada et al. 1991, J. Pharm. Pharmacol. 43: 605-608 disclosesustained-release drug conjugates with lactic acid oligomers.

[0109] Specifically, polymer-conjugated drugs have been reported in theprior art, and attempts to adapt particulate conjugates have also beenreported.

[0110] Ryser et al., U.S. Pat. No. 4,847,240, issued Jul. 11, 1989,provides cationic polymers for conjugation to compounds that are poorlytransported into cells. Examples include the antineoplastic drugmethotrexate conjugated with polylysine and other polycationic aminoacids as carriers.

[0111] Ellestad et al., U.S. Pat. No. 5,053,394, issued Oct. 1, 1991,provides carrier-drug conjugates of methyltrithiol antibacterial andantitumor agents with a spacer linked to a targeting molecule that is anantibody or fragment thereof, growth factors or steroids.

[0112] Kopecek et al., U.S. Pat. No. 5,258,453, issued Nov. 2, 1993,provides antitumor compositions comprising both an anticancer drug and aphotoactivatable drug attached to a copolymeric carrier by functionalgroups labile in cellular lysosomes, optionally containing a targetingmoiety that are monoclonal antibodies, hormones, etc.

[0113] Yatvin et al., U.S. Pat. No. 5,543,390, issued Aug. 6, 1996,discloses microparticles conjugated to antiproliferative drugs.

[0114] Yatvin et al., U.S. Pat. No. 5,543,391, issued Aug. 6, 1996,discloses microparticles conjugated to antiproliferative drugs.

[0115] Negre et al., 1992, Antimicrob. Agents and Chemother. 36:2228-2232 disclose the use of neutral mannose-substituted polylysineconjugates with an anti-leischmanial drug (allopurinol riboside) totreat murine infected macrophages in vitro.

[0116] Yatvin, 1991, Select. Cancer. Therapeut. 7: 23-28 discusses theuse of particulate carriers for drug targeting.

[0117] Hunter et al., 1988, J. Pharm. Pharmacol. 40: 161-165 discloseliposome-mediated delivery of anti-leischmanial drugs to infected murinemacrophages in vitro.

[0118] Saffran et al., 1986, Science 233: 1081-1084 disclose drugrelease from a particulate carrier in the gut resulting from degradationof the carrier by enzymes produced by intestinal microflora.

[0119] A particular human disease related to infection of phagocyticcells by a microorganism is tuberculosis, caused by infection withMycobacterium tuberculosis. This disease typically arises afterinhalation in phagocytic macrophages in the lung, where characteristiclocalized sites of infection (termed tubercules) are formed and comprisesites of further systemic infection. Although previously well-controlledby antibiotics such as isoniazid, the development of drug-resistance bythe infectious agent, and the increased numbers of immune-compromisedindividuals with the outbreak of the AIDS crisis has created a nearepidemic of tuberculosis cases world-wide. In 1997, the World HealthOrganization reported tuberculosis to be the world's top infectiouskiller.

[0120] About one-third of new tuberculosis cases are resistant to thecurrent drug-treatment regimes. It is estimated that drug-resistanttuberculosis accounts for between 2% and 14% of total tuberculosis casesworldwide. As tuberculosis is spread by air-borne droplets from coughingby infected individuals, and its spread is further facilitated incrowded environments such as cities, there is a great potential for aprecipitous increase in tuberculosis infections that will not be easilycontrolled by conventional medicinal intervention such as isoniazidadministration. Lethal strains of tuberculosis have the potential forrapid spread, since only about one in ten patients receives the medicaltreatment necessary to contain and successfully treat the disease. Thus,there exists in this art a need to develop new and better treatments fortuberculosis, particularly tuberculosis infections resistant totraditional antibiotic treatments.

SUMMARY OF THE INVENTION

[0121] The present invention is directed to improved reagents andmethods for delivering antibiotic, antimicrobial or antiviral compounds,drugs or agents to phagocytic cells in vivo and in vitro. In particular,the invention is directed towards delivery of antimicrobial compounds,drugs and agents specific for treatment of tuberculosis and otherMycobacterium-caused diseases in humans.

[0122] The invention provides drug delivery vehicles that aremicroparticles conjugated to, coated with, or impregnated with one or amultiplicity of antimicrobial compounds, drugs or agents specific forthe treatment of tuberculosis and other Mycobacterium-caused diseases inanimals, most preferably humans. In one preferred embodiment, theantibiotic, antimicrobial or antiviral compound, drug or agent is aprodrug of an activated form of the anti-tuberculosis drug isoniazid. Ina second preferred embodiment, the antibiotic, antimicrobial orantiviral compound, drug or agent is a competitive inhibitor of longchain enol-acyl carrier protein reductase (termed InhA), an M.tuberculosis-encoded enzyme required for production of an essentialbacterial cell wall component, mycolic acid. In a third preferredembodiment, the antibiotic, antimicrobial or antiviral compound, drug oragent is an irreversible inhibitor of InhA, otherwise termed a “suicidesubstrate” herein.

[0123] In one aspect, this delivery system achieves specific delivery ofantibiotic, antimicrobial or antiviral compounds, drugs or agents tophagocytic cells through conjugating the antibiotic, antimicrobial orantiviral compound, drug or agent with a particular microparticle via acleavable linker moiety that is specifically cleaved in an infectedcell. Alternatively, specific delivery is achieved by impregnating theantibiotic, antimicrobial or antiviral compound, drug or agent into aporous microparticle, which is then coated with a specifically-degradedcoating material that is specifically degraded in an infected cell. Inyet another alternative embodiment, the delivery system comprises anonporous microparticle wherein an antibiotic, antiviral andantimicrobial compound, drug or agent is prepared as a coating on theparticle surface, and the particle is then further coated by aspecifically-degradable coating material that is specifically degradedin an infected cell. In another embodiment, a porous or non-porousmicroparticle is impregnated or coated with a first antibiotic,antimicrobial or antiviral compound, drug or agent, then coated with aspecifically-degradable or non-specifically degradable coating material,then further coated with a second coating of a antibiotic, antimicrobialor antiviral compound, drug or agent that can be the same or differentthan the first coating of antibiotic, antimicrobial or antiviralcompound, drug or agent, then further coated with a second coating of aspecifically-degradable or non-specifically degradable coating materialthat may be the same or different than the first specifically-degradableor non-specifically degradable coating, wherein the microparticle cancomprise a multiplicity of such alternating coatings of antibiotic,antimicrobial or antiviral compounds, drugs and agents andspecifically-degradable or non-specifically degradable coatings,provided that the final coating of the microparticle is aspecifically-degradable coating that is specifically degraded only in acell infected with a pathological or disease-causing microorganism, mostpreferably a Mycobacterium species. In each embodiment of themicroparticles of the invention, specific release of the antibiotic,antimicrobial or antiviral compounds, drugs and agents from themicroparticle is achieved by enzymatic or chemical release of thecompound, drug or agent from the microparticle by cleavage of thecleavable linker moiety or the specifically-degradable coating materialin infected phagocytic cells. Such microparticles can be produced toprovide sequential, delayed, sustained or controlled release of theantibiotic, antimicrobial or antiviral compounds, drugs or agents of theinvention.

[0124] In a first aspect, the specific delivery of antibiotic,antimicrobial or antiviral compounds, drugs or agents achieved by thepresent invention results from conjugating, impregnating or coating suchcompounds, drugs or agents to microparticles. Specific intracellularaccumulation and facilitated cell entry is mediated by the phagocyticuptake of microparticle-conjugated antibiotic, antimicrobial orantiviral compounds, drugs or agents by such cells. Preferredembodiments of phagocytic cellular targets include phagocytichematopoietic cells, preferably macrophages and phagocytic neutrophiles,most preferably macrophages, mononuclear cells and phagocyticneutrophiles from lung tissue.

[0125] Particularly preferred targets of the microparticle-conjugatedantibiotic, antimicrobial or antiviral compounds, drugs or agents of theinvention are phagocytic cells, including phagocytic hematopoieticcells, preferably macrophages and phagocytic neutrophiles and mostpreferably macrophages, mononuclear cells and phagocytic neutrophilesfrom lung tissue that are infected with M. tuberculosis, M. africanum,M. bovis or any other microorganism that causes tuberculosis in ananimal, most preferably a human. Also preferred targets are cellsinfected with M. leprae, M. avium, M. intracellulare, M. scrofulaceum,M. kansasii, M. xenopi, M. marinum, M. ulcerans, M. fortuitum and M.chelonae. For such cells, the embodiments of themicroparticle-conjugated antibiotic, antimicrobial or antiviralcompounds, drugs or agents of the invention are comprised of cleavablelinker moieties or specifically-degradable coatings whereby chemical orenzymatic cleavage of said linker moieties or coatings is specific fortuberculosis- or other disease-causing Mycobacterium-infected phagocyticcells. Such microparticles provide for infected cell-specific release ofantibiotic, antimicrobial or antiviral compounds, drugs or agents, suchas isoniazid, activated isoniazid, rifampin, streptomycin, ethambutoland pyrazinamide, and competitive, non-competitive and “suicidesubstrate” InhA inhibitors or any other anti-tuberculosis oranti-Mycobacterium drug or agent, in such infected cells. It isunderstood that all phagocytic cells are expected to take up suchmicroparticle-conjugated or coated antibiotic, antimicrobial orantiviral embodiments of the invention. However, it is an advantageousfeature of the microparticle-conjugated antibiotic, antimicrobial orantiviral compounds of the invention that specific release ofbiologically-active forms of such antibiotic, antimicrobial or antiviraldrugs or agents is dependent on the presence of the infectiousmicroorganism in the phagocytic cell.

[0126] The invention provides compositions of matter and pharmaceuticalcompositions thereof comprising a porous microparticle into which isimpregnated with an antibiotic, antimicrobial or antiviral compound, theimpregnated porous microparticle being further coated with aspecifically-degradable coating material. In this aspect of theinvention, the specifically-degradable coating material is specificallydegraded inside a phagocytic mammalian cell infected with atuberculosis-causing or other Mycobacterium-associated disease-causingmicroorganism, allowing the specific release of the antibiotic,antimicrobial or antiviral compound within the infected cell. Inpreferred embodiments, the specifically-degradable coating material is asubstrate for a protein having an enzymatic activity found specificallyin phagocytic cells infected with a tuberculosis-causing or otherMycobacterium-associated disease-causing microorganism. In additionalpreferred embodiments, the specifically-degraded coating material ischemically cleaved under physiological conditions that are specific forphagocytic cells infected with a tuberculosis-causing microorganism. Inpreferred embodiments, the antibiotic, antimicrobial or antiviralcompound, drug or agent impregnating the microparticle is an activatedembodiment of said compound, drug or agent, as defined herein. Inalternative embodiments, the microparticle is impregnated with amultiplicity of antibiotic, antimicrobial or antiviral compounds, drugsor agents.

[0127] In alternative aspects, the coating material is nonspecificallycleaved chemically or enzymatically inside a phagocytic cell, whereinthe antibiotic, antimicrobial or antiviral compound, drug or agent is ina form that is only specifically activated in the cell when the cell isinfected with a tuberculosis-causing or other Mycobacterium-associateddisease-causing microorganism (wherein said antibiotic, antimicrobial orantiviral compounds, drugs or agents are termed “prodrugs” as definedherein when provided in this form). In alternative embodiments, themicroparticle is impregnated with a multiplicity of antibiotic,antimicrobial or antiviral compounds, drugs or agents or prodrugembodiments thereof

[0128] In preferred embodiments of the invention, the antibioticcompound is a specifically bactericidal or bacteriostatic against amicroorganism that causes tuberculosis in an animal, most preferably ahuman, most preferably M. tuberculosis, M. africanum, M. bovis.Preferred antibiotic compounds used to impregnate such porousmicroparticles include activated isoniazid, rifampin, streptomycin,ethambutol and pyrazinamide, and competitive, uncompetitive,non-competitive and “suicide substrate” InhA inhibitors or any otheranti-tuberculosis or anti-Mycobacteriurn compound, drug or agent.Activated and prodrug embodiments of these or other antibiotic,antimicrobial or antiviral compounds, drugs or agents are alsopreferred, and activated embodiments of said drugs are particularlypreferred.

[0129] In preferred embodiments, the antimycobacterial drugs used in thepractice of the invention are “activated” embodiments (as definedherein) of competitive, uncompetitive, non-competitive and “suicidesubstrate” inhibitors of long chain enol-acyl carrier protein reductase(InhA), a Mycobacterium-specific enzyme necessary for the production ofmycolic acid, which an essential component of the mycobacterial cellwall. Inhibition of this enzyme by isoniazid is the basis of currentanti-tuberculosis treatment modalities, and resistance to isoniazid isthe principle form of drug resistance exhibited by mycobacteria. Thecompounds of the invention overcome resistance by being “pre-activated”,i.e., these compounds do not rely on activation in themycobacterium-infected cell for activity (unlike compounds do not relyon activation in the mycobacterium-infected cell for activity (unlikeisoniazid itself). Thus, it is expected that resistance is less likelyto be developed against these drugs. In a preferred embodiment, thesecompounds have the generic structure:

[0130] wherein X can be C or O; Y can be N or C; R1 and R2 can each beindependently an electron pair, H, CH₃, CH₂—CH₃, or O(CH₂)₃O or togethercan be ═O, ═CH₂, —CH₂—CH₂—, ═CH—CH═CH₂,═CH—COOCH₂—CH₃, or OCH₂.

[0131] The invention also provides compositions of matter andpharmaceutical compositions thereof comprising a nonporous microparticleonto which is coated an antibiotic, antimicrobial or antiviral compound,the coated nonporous microparticle being further coated with aspecifically-degradable coating material. In this aspect of theinvention, the specifically-degradable coating material is specificallydegraded inside a phagocytic mammalian cell infected with atuberculosis-causing or other Mycobacterium-associated disease-causingmicroorganism, allowing the specific release of the antibiotic,antimicrobial or antiviral compound within the infected cell. Inpreferred embodiments, the specifically-degradable coating material is asubstrate for a protein having an enzymatic activity found specificallyin phagocytic cells infected with a tuberculosis-causing or otherMycobacterium-associated disease-causing microorganism. In additionalpreferred embodiments, the specifically-degraded coating material ischemically cleaved under physiological conditions that are specific forphagocytic cells infected with a tuberculosis-causing microorganism. Inpreferred embodiments, the antibiotic, antimicrobial or antiviralcompound, drug or agent coating the microparticle is an activatedembodiment of said compound, drug or agent, as defined herein. Inalternative aspects, the coating material is nonspecifically cleavedchemically or enzymatically inside a phagocytic cell, wherein theantibiotic, antimicrobial or antiviral compound, drug or agent is in aform that is only specifically activated in the cell when the cell isinfected with a tuberculosis-causing or other Mycobacterium-associateddisease-causing microorganism (wherein said antibiotic, antimicrobial orantiviral compound, drug or agent is termed a “prodrug” as definedherein when provided in this form). In alternative embodiments, themicroparticle is coated with a multiplicity of antibiotic, antimicrobialor antiviral compounds, drugs or agents or prodrug embodiments thereof.

[0132] In preferred embodiments of the invention, the antibioticcompound is a specifically bactericidal or bacteriostatic against amicroorganism that causes tuberculosis in an animal, most preferably ahuman, most preferably M. tuberculosis, M. africanum, M. bovis.Preferred antibiotic compounds used to coat such porous microparticlesinclude activated isoniazid, rifampin, streptomycin, ethambutol andpyrazinamide, and competitive, uncompetitive, non-competitive and“suicide substrate” InhA inhibitors or any other anti-tuberculosis oranti-Mycobacterium compound, drug or agent. Activated and prodrugembodiments of these or other antibiotic, antimicrobial or antiviralcompounds, drugs or agents are also preferred, and activated embodimentsare particularly preferred. Most preferred embodiments have the genericstructure disclosed above

[0133] Additional embodiments of the compositions of matter andpharmaceutical compositions thereof comprising the porous andnon-porous, impregnated or coated microparticles of the invention areprovided wherein the porous or non-porous microparticle is impregnatedor coated with a first antibiotic, antimicrobial or antiviral compound,drug or agent, then coated with a specifically-degradable ornon-specifically degradable coating material, then further coated with asecond coating of a antibiotic, antimicrobial or antiviral compound,drug or agent that can be the same or different than the first coatingof antibiotic, antimicrobial or antiviral compound, drug or agent, thenfurther coated with a second coating of a specifically-degradable ornon-specifically degradable coating material that may be the same ordifferent than the first specifically-degradable or non-specificallydegradable coating, wherein the microparticle can comprise amultiplicity of such alternating coatings of antibiotic, antimicrobialor antiviral compounds, drugs or agents and specifically-degradable ornon-specifically degradable coatings, provided that the final coating ofthe microparticle is a specifically-degradable coating that isspecifically degraded in a cell infected with a pathological ordisease-causing microorganism, most preferably a Mycobacterium species.Such microparticles can be produced to provide sequential, delayed,sustained or controlled release of the antibiotic, antimicrobial orantiviral compounds, drugs or agents of the invention. In eachembodiment of the microparticles of the invention, specific release ofthe antibiotic, antimicrobial or antiviral compound, drug or agent fromthe microparticle is achieved by enzymatic or chemical release of thecompound, drug or agent from the microparticle by cleavage of thespecifically-degradable coating material in infected phagocytic cells.Antibiotic, antimicrobial and antiviral compounds, drugs or agentsreleased by non-specific chemical or enzymatic degradation areadvantageously provided in inactive, prodrug forms that are specificallyactivated in cells infected with pathological or disease-causingmicroorganism, most preferably a Mycobacterium species. In onealternative embodiment of this aspect of the invention, the “gatekeeper”for release of the antibiotic, antimicrobial or antiviral drug, compoundor agent coating the microparticle is the ultimate,specifically-degraded coating material, which is only removed from themicroparticle in a phagocytic cell infected with a pathological ordisease-causing microorganism, most preferably a Mycobacterium species.In preferred embodiments, the antibiotic, antimicrobial or antiviralagent is provided in an activated form as defined herein. In saidpreferred embodiments, the “gatekeeper” specifically-degraded coatingmaterial prevents release of physiologically-significant amounts of theactivated compound, drug or agent anywhere other than inside an infectedphagocytic cell, most preferably a phagocytic cell infected with apathological or disease-causing microorganism, most preferably aMycobacterium species.

[0134] In an alternative embodiment, each antimicrobial, antibiotic orantiviral drug, compound or agent is provided in the form of a prodrugthat is activated only in a phagocytic cell infected with saidpathological or disease-causing microorganism, most preferably aMycobacterium species. In this alternative embodiment, delivery of theantibacterial, antibiotic or antiviral drug, compound or agent in anactive form to a phagocytic cell will only occur in such a cell that isinfected with a pathological or disease-causing microorganism, mostpreferably a Mycobacterium species wherein both thespecifically-degradable coating and the prodrug are degraded andactivated, respectively, by an enzymatic or chemical reaction specificfor the infected cell.

[0135] In these aspects of the invention, the antibiotic, antimicrobialor antiviral compound, drug or agent will be understood to dissolve fromthe surface of the microparticle upon enzymatic or chemical degradationof the organic coating material. Release of the antibiotic,antimicrobial or antiviral compound, drug or agent can be accomplishedsimply be mass action, i.e., whereby the compound dissolves from thesurface of the nonporous microparticle into the surrounding cytoplasmwithin the cell, or leaches or is released from the porousmicroparticle.

[0136] The invention also provides compositions of matter andpharmaceutical compositions thereof comprising an antibiotic,antimicrobial or antiviral compound, drug or agent linked to amicroparticle via a cleavable linker moiety. The cleavable linkermoieties of the invention comprise two linker functional groups, whereinthe cleavable linker moiety has a first end and a second end. Themicroparticle is attached to the first end of the cleavable linkermoiety through a first linker functional group and the antibiotic,antimicrobial or antiviral compound, drug or agent is attached to thesecond end of the cleavable linker moiety through a second linkerfunctional group. The cleavable linker moieties of the invention arespecifically cleaved inside an infected phagocytic mammalian cell, forexample, a phagocytic cell infected with a tuberculosis-causing or otherMycobacterium-associated disease-causing microorganism. In preferredembodiments, the cleavable linker moieties of the invention comprise asubstrate for a protein having an enzymatic activity found specificallyin phagocytic cells infected with a tuberculosis-causing or otherMycobacterium-associated disease-causing microorganism. In a particularembodiment of this aspect of the invention, the cleavable linker moietyis a peptide of formula (amino acid)_(n), wherein n is an integerbetween 2 and 100, preferably wherein the peptide comprises a polymer ofone or more amino acids. In additional preferred embodiments, thecleavable linker moieties of the invention are moieties that arechemically cleaved under physiological conditions that are specific forphagocytic cells infected with a tuberculosis-causing microorganism. Inpreferred embodiments, the antibiotic, antimicrobial or antiviralcompound, drug or agent impregnating the microparticle is an activatedembodiment of said compound, drug or agent, as defined herein. Inalternative embodiments, the microparticles of the invention areprovided comprising either a multiplicity of antimicrobial, antibioticor antiviral compounds, drugs, or agents or a multiplicity of cleavablelinker moieties, or both.

[0137] In alternative aspects, the cleavable linker moieties arenonspecifically cleaved chemically or enzymatically inside a phagocyticcell, wherein the antibiotic, antimicrobial or antiviral compound, drugor agent is in a form that is only specifically activated in the cellwhen the cell is infected with a tuberculosis-causing or otherMycobacterium-associated disease-causing microorganism (wherein saidantibiotic, antimicrobial or antiviral compounds, drugs or agents aretermed “prodrugs” as defined herein when provided in this form).

[0138] In preferred embodiments of the invention, the antibioticcompound is a specifically bactericidal or bacteriostatic against amicroorganism that causes tuberculosis in an animal, most preferably ahuman, most preferably M. tuberculosis, M. africanum, M. bovis. Inpreferred embodiments, the antibiotic compound is isoniazid, activatedisoniazid, rifampin, streptomycin, ethambutol and pyrazinamide, andcompetitive, uncompetitive, non-competitive and “suicide substrate” InhAinhibitors or any other anti-tuberculosis or anti-Mycobacteriumcompound, drug or agent. Activated and prodrug embodiments of these orother antibiotic, antimicrobial or antiviral compounds, drugs or agentsare also preferred.

[0139] The most preferred embodiments of the microparticles of theinvention comprise prodrugs forms of activated isoniazid conjugates withNAD (termed isoniazid-NAD analogues, of INA, herein) that areinactivated by covalent modification of the activated drug to blockbinding of the drug to NAD-requiring enzymes, including InhA andmammalian cell, most preferably human cell-derived, NAD requiringenzymes. In the most preferred embodiments of this aspect of theinvention, the inactivated prodrug form is specifically activated onlyin Mycobacterium-infected cells. In one aspect, such specific cleavageis due to a chemical linkage in the derivative that is labile within theinfected cell due to conditions caused by or that result from infectionof the cell with the mycobacteria. In another preferred aspect, suchspecific cleavage is due to an enzymatic activity which is producedeither by the mycobacteria itself or by the cell as the result ofinfection with said mycobacteria, wherein the linkage is enzymaticallycleaved by the enzymatic activity. In particularly preferredembodiments, the derivatizing group is a urea moiety that isspecifically cleaved in Mycobacteria-infected cells by amycobacteria-encoded urease.

[0140] The microparticle-drug conjugates of this invention have numerousadvantages. First, the drug-microparticle conjugates are specificallytaken up by phagocytic mammalian cells. Second, antibiotic,antimicrobial or antiviral compound, drugs or agents, most preferablyanti-tuberculosis and anti-Mycobacterium compounds, drugs or agentscomprising the drug-microparticle conjugates of the invention, arelinked to the microparticle or covered by a coating comprising aspecifically degradable moiety or material that is specifically cleavedupon entry into appropriate phagocytic cells, i.e., phagocytic cellsinfected with a tuberculosis-causing or other Mycobacterium-associateddisease-causing microorganism. Third, the conjugates of the inventioncan be combined with other drug delivery approaches to further increasespecificity and to take advantage of useful advances in the art. Fourth,the specificity of microparticle delivery to phagocytic cells and thespecificity of conjugated antibiotic, antimicrobial or antiviralcompound, drug or agent release in infected phagocytic cells permits theuse and administration of efficacious antibiotic, antimicrobial orantiviral compounds, drugs or agents that are otherwise too toxic to beadministered directly to an animal. Fifth, the specific delivery of themicroparticles of the invention to phagocytic cells, and the specificrelease of antibiotic, antimicrobial or antiviral compounds, drugs oragents, and particularly activated embodiments thereof, permits directadministration of forms of said compounds, drugs and agents as they areactivated by infectious organism-specific enzymatic or chemicalmodification, thereby providing a way of overcoming common forms ofresistance to otherwise or previously efficacious antibiotic,antimicrobial or antiviral compounds, drugs or agents.

[0141] Thus, the invention also provides a method of killing amicroorganism infecting a mammalian cell, preferably a phagocyticmammalian cell. This method comprises contacting an infected phagocyticmammalian cell with the compositions of matter or pharmaceuticalcompositions of the invention in vivo or in vitro. The invention alsoprovides methods for treating microbial infections in an animal, mostpreferably a human wherein the infecting microbe is present inside aphagocytic cell in the human, the method comprising administering atherapeutically effective amount of the compositions of matter orpharmaceutical compositions of the invention to the human in apharmaceutically acceptable carrier. Thus, the invention also providespharmaceutical compositions comprising the compositions of matter of theinvention in a pharmaceutically acceptable carrier. In most preferredembodiments, the infecting microorganism is a tuberculosis-causingmicroorganism such as M. tuberculosis, M. africanum or M. bovis.

[0142] Thus, in a first aspect the invention provides compositions ofmatter, pharmaceutical compositions and methods for targetingantibiotic, antimicrobial or antiviral compounds, drugs and agents tophagocytic cells. In a second aspect, the invention providescompositions of matter, pharmaceutical compositions and methods for thespecific release of antibiotic, antimicrobial or antiviral compounds,drugs and agents inside phagocytic cells. The invention in yet a thirdaspect provides compositions of matter, pharmaceutical compositions andmethods for intracellular delivery of targeted antibiotic, antimicrobialor antiviral compounds, drugs and agents to phagocytic cells. In each ofthese aspects is provided compositions of matter, pharmaceuticalcompositions and methods for introducing antibiotic, antimicrobial orantiviral compounds, drugs and agents into phagocytic mammalian cellswherein the unconjugated compound, drug or agent would not otherwiseenter said phagocytic cell, the compound, drug or agent would not bespecifically targeted to said phagocytic cell or the compound, drug oragent would have deleterious or toxic effects on non-infected cells. Inthis aspect is included the introduction of said compounds, drugs oragents in antibiotic, antimicrobial or antiviral embodiments that wouldnot otherwise enter the cell, for example, as charged embodiments orsalts, or wherein the compound, drug or agent is unstable or has a shorthalf-life. In addition, the antibiotic, antimicrobial or antiviralcompounds, drugs and agents useful in this invention are provided inactivated forms in which they are toxic to normal cells, or which areactivated by infectious agent-specific enzymatic or chemicalmodifications, but which are conjugated to or coated within amicroparticle of the invention and released only in phagocytic cellsinfected with a tuberculosis or other Mycobacterium-associateddisease-causing microorganism In yet another aspect is providedcompositions of matter, pharmaceutical compositions and methods for thespecific coordinated targeting of more than one antibiotic,antimicrobial or antiviral compound to infected phagocytic mammaliancells. In another aspect, the invention provides compositions of matter,pharmaceutical compositions and methods for the introduction andspecific release of antibiotic, antimicrobial or antiviral compounds,drugs or agents, preferably anti-tuberculosis and anti-Mycobacteriumcompounds, drugs or agents, and other compounds into cells infected by atuberculosis-causing or other Mycobacterium-associated disease-causingpathological microorganism. In a final aspect, the invention providescompositions of matter, pharmaceutical compositions and methods forsequential, delayed, sustained or controlled intracellular release ofantibiotic, antimicrobial, or antiviral compounds, drugs or agentsimpregnated within a coated, porous microparticle, or coated onto anonporous microparticle, wherein the degradation of either a layer ofthe coating or the microparticle or both provides said sequential,delayed, sustained or controlled intracellular release of theantibiotic, antimicrobial or antiviral compounds, drugs or agents of theinvention.

[0143] Specific preferred embodiments of the present invention willbecome evident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0144]FIG. 1 is a diagram showing activation of the anti-tuberculosisdrug isoniazid.

[0145]FIG. 2 is a schematic representation of the practice of theinvention.

[0146]FIG. 3 depicts the synthetic scheme put forth in Example 1.

[0147]FIG. 4 shows a synthetic scheme for producing a soluble dye-linkedcompound as set forth in Example 1.

[0148]FIG. 5 depicts competitive and suicide substrates of InhA.

[0149]FIG. 6 depicts INA inactivated by covalent modification with aurea moiety.

[0150]FIG. 7 is a schematic diagram of urease-catalyzed cleavage of thecarbamate-containing polymer described in Example 9.

[0151]FIG. 8 shows a synthetic scheme for producing a urease-catalyzedcleavage of the carbamate-containing polymer described in Example 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0152] The present invention provides compositions of matter and methodsfor facilitating the entry of antibiotic, antimicrobial or antiviralcompounds, drugs or agents into phagocytic cells. For the purposes ofthis invention, the term “antibiotic, antimicrobial or antiviralcompounds, drugs or agents” is intended to encompass allnaturally-occurring or synthetic chemical compounds capable of having atoxic, cytocidal or cytostatic effect on pathogenic or disease-causingmicroorganisms, most preferably tuberculosis-causing microorganismsincluding but not limited to M. tuberculosis, M. africanum, M. bovis, M.leprae, M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M.xenopi, M. marinum, M. ulcerans, M. fortuitum and M. chelonae. Thesecompounds are intended to include but are not limited to all varietiesof drugs or agents, particularly antibiotic and antimicrobial drugs, andmost preferably anti-tuberculosis drugs and agents, including but notlimited to isoniazid, activated isoniazid, rifampin, streptomycin,ethambutol, pyrazinamide, and competitive, uncompetitive,non-competitive and “suicide substrate” InhA inhibitors or any otheranti-tuberculosis or anti-Mycobacterium compound, drug or agent.

[0153] In preferred embodiments, the antibiotic, antimicrobial orantiviral compound, drug or agent coating the microparticle is anactivated embodiment of said compound, drug or agent, as defined herein,wherein said activated embodiment is not otherwise routinelyadministered to an animal because it is toxic to non-infected cells. Theterms “activated” and “activated form” of antibiotic, antimicrobial orantiviral compounds, drugs or agents as provided herein are intended toencompass embodiments of said compounds, drugs or agents that are toxicto both infected and uninfected cells. These terms include embodimentswherein the compound, drug or agent is in a form, for example, that isthe result of an infectious microbe-specific modification of theunactivated form of the compound, drug or agent. These terms alsoencompass compounds that have been enzymatically or chemically modifiedin an infected cell and have antibiotic, antimicrobial or antiviralproperties conferred or enhanced thereby. In the latter instance, theterms in particular are related to embodiments whereby the pathologicalor disease-causing microorganism has developed resistance to thecompounds, drugs or agents by attenuation, mutation or ablation of thechemical or enzymatic activity in the infected cell. Introducing theactivated form of the compound, drug or agent directly into the infectedphagocytic cell provides a route for overcoming these types ofresistance. In this way, the invention provides a way of delivering theactivated compound, drug or agent to the infectious microbe, even in theevent that the microbe is resistant to the compound, drug or agentbecause it no longer efficiently effects said modification of theunactivated form of the compound drug or agent, whether by mutation,loss or attenuation of gene expression, selection or otherwise.

[0154] An illustration of this type of activation is shown in FIG. 1. Inthe Figure, the anti-tuberculosis drug isoniazid is gently oxidized inthe presence of divalent manganese ion and hydrogen peroxide to providethe isonicotinic acyl anion. (This reaction is preferably performed inan aprotic solvent to stabilize the anion.) Under slightly basicconditions (pH 7.5) the anion reacts with the oxidized form ofnicotinamide adenine dinucleotide (NAD+) to form isoniazid-NAD analogue,or INA as described herein, that inhibits long chain enol-acyl carrierprotein reductase (InhA), an enzyme needed for synthesis of mycolicacid, a critical component of the M. tuberculosis cell wall. Inhibitionof mycolic acid production is the molecular basis of isoniazidanti-tuberculosis activity.

[0155] In a second illustrative embodiment, an NAD analogue thatinhibits InhA, most preferably INA, is derivatized by covalently linkinga urea moiety at a position in the molecule involved in binding themolecule with NAD-requiring enzymes, including mammalian NAD-requiringenzymes and InhA. The activated isoniazid-NAD analogues of the inventionare provided in a form that is inactive in a mammalian, most preferablya human, cell not infected with mycobacteria. In these embodiments, theanalogue, most preferably INA, is derivatized at a conserved position inthe NAD molecule involved in NAD binding to NAD-requiring enzymes. Mostpreferably, such positions include but are not limited to the formamidegroup of the pyridine portion of the NAD component of INA, and the1-amino group of the adenine portion of the NAD component of INA.Derivatives containing blocking groups at these positions are severelyinhibited (by at least about 10-fold in binding affinity) in binding toNAD-requiring enzymes.

[0156] Delivery of these prodrugs to phagocytic cells is achieved usingthe microparticles of the invention. Specific activation of thederivatized prodrugs is achieved only in Mycobacteria-infected cells,because only those cells produce the conditions (chemical or enzymatic)under which the prodrug is turned into the active form of the drug. Inpreferred embodiments, the prodrug is a urea-derivatized form of INA,which is specifically activated to INA in Mycobactertium-infected cellsby a specific urease made in those cells and not in any other mammalian,more preferably human, and most preferably phagocytic cells.

[0157] This invention provides microparticle-linked antibiotic,antimicrobial or antiviral compounds, drugs or agents wherein anantimicrobial drug, more preferably an anti-tuberculosis drug oranti-Mycobacterium drug is linked to a microparticle via a cleavablelinker moiety, is impregnated within a porous microparticle or is coatedonto a nonporous microparticle wherein said impregnated or coatedmicroparticle is further coated with a specifically-degradable coatingmaterial. The microparticle-linked antibiotic, antimicrobial orantiviral compounds, drugs, or agents are taken up by phagocytic cells,most preferably hematopoietic phagocytic cells such as monocytes andmacrophages, and the compounds, drugs and agents are specificallyreleased in such phagocytic cells that are infected with a microorganismthat produces (or whereby infection with the microorganism produces inthe cell) an enzymatic or chemical activity that specifically cleavesthe cleavable linker moiety or the specifically-degradable coatingmaterial. The practice of the invention is schematically represented inFIG. 2.

[0158] The term “anti-tuberculosis drug or anti-Mycobacterium drug” isintended to encompass any pharmacological agent effective in inhibiting,attenuating, combating or overcoming infection of phagocytic mammaliancells by a tuberculosis-causing or other disease-causing Mycobacteriumspecies microbial pathogen in vivo or in vitro. Anti-tuberculosis drugsas provided by the invention include but are not limited to isoniazid,activated isoniazid, rifampin, capreomycin, ethionamide, cycloserine,ciprofloxacin, amikacin, streptomycin, ethambutol, pyrazinamide, andcompetitive, uncompetitive, non-competitive and “suicide substrate” InhAinhibitors as disclosed herein. Activated and prodrug embodiments ofthese or other antibiotic, antimicrobial or antiviral compounds, drugsor agents are also preferred. In preferred embodiments, theanti-mycobacterial drug is a drug that inhibits InhA, most preferably anactivated form of isoniazid identified as isoniazid-NAD analogue andrelated compounds, that is provided as a prodrug as described herein.More preferably, the prodrug form of said InhA-inhibiting activatedisoniazid derivatives is inactivated by covalent modification of themolecule at a site involved in binding to an NAD-requiring enzyme. Evenmore preferably, the modification is a modification that is specificallycleaved only in a mammalian cell, more preferably a human cell and mostpreferably a phagocytic cell that is infected with a Mycobacteriumspecies. Most preferably, the covalent modification is attachment of aurea moiety to the formamide group of the nicotinamide portion of INA orto the 1-amino group of the adenine portion of INA, wherein said ureamoiety is specifically cleaved in an infected cell by a urease producedby the infecting Mycobacterium species.

[0159] This invention provides microparticle-linked antibiotic,antimicrobial or antiviral compounds, drugs or agents for specific celltargeting to phagocytic mammalian cells. As used herein, the term“phagocytic mammalian cells” is intended to encompass but is not limitedto monocytes, macrophages, peritoneal macrophages, alveolar macrophages,Kuppfer cells of the liver, macrophage cells resident in the centralnervous system and the skin, all tissue inflammatory and noninflammatorymacrophages, and phagocytic bone marrow cells, and most preferablyalveolar macrophages.

[0160] In the antibiotic, antimicrobial and antiviral compounds, drugsor agents as provided by this invention, said antibiotic, antimicrobialor antiviral compounds, drugs or agents, most preferablyanti-tuberculosis and anti-Mycobacterium compounds, drugs or agents, arelinked to, impregnated into or coated onto microparticles that arespecifically phagocytized by phagocytic mammalian cells. It is anadvantage of the present invention that said antibiotic, antimicrobialand antiviral compounds, drugs or agents are specifically targeted tophagocytic mammalian cells, including, inter alia, monocytes andmacrophages as described herein, via the microparticles that are acomponent of the antibiotic, antimicrobial and antiviral compounds,drugs or agents of the invention.

[0161] The term “microparticle” as used herein is intended to encompassany particulate bead, sphere, particle or carrier, whether biodegradableor nonbiodegradable, comprised of naturally-occurring or synthetic,organic or inorganic materials that is specifically phagocytized byphagocytic mammalian cells. In particular, the microparticle componentof the antibiotic, antimicrobial or antiviral compounds, drugs or agentsof the invention include any particulate bead, sphere, particle orcarrier having a diameter of about 1 to about 5000 nanometers (about0.001-5 μm), more preferably 1-5 μm in diameter. The microparticles ofthe invention are provided comprised of polystyrene, cellulose, silica,polyacrylamide, and various polysaccharides including dextran, agarose,cellulose and modified, crosslinked and derivatized embodiments thereof.Specific examples of the microparticles of the invention includepolystyrene, cellulose, dextran crosslinked with epichlorohydrin(Sephadex™, Pharmacia, Uppsala, Sweden), polyacrylamide crosslinked withbisacrylamide (Biogel™, BioRad, U.S.A.), agar, glass beads and latexbeads. Derivatized microparticles include microparticles derivatizedwith carboxyalkyl groups such as carboxymethyl, phosphoryl andsubstituted phosphoryl groups, sulfate, sulfhydryl and sulfonyl groups,and amino and substituted amino groups.

[0162] In one embodiment of the invention, said microparticle is aporous particle having a defined degree of porosity and comprised ofpores having a defined size range, wherein the antibiotic, antimicrobialor antiviral compounds, drugs or agents are impregnated within the poresof the microparticle. In such embodiments, a chemically orenzymatically-degradable coating covers the surface or outside extent ofthe microparticle, wherein the coating is specifically chemically orenzymatically degraded within the particular infected phagocytic cellafter phagocytosis and is not degraded systemically or in uninfectedphagocytic cells. In preferred embodiments, the phagocytic cell isinfected with a tuberculosis-causing or other Mycobacterium-associateddisease-causing microorganism. In alternative embodiments, the porousmicroparticle is impregnated with a multiplicity of antibiotic,antimicrobial or antiviral compounds, drugs or agents.

[0163] In a second embodiment of the invention, the microparticle iseither a porous or a nonporous particle. In such embodiments, thesurface or outside extent of the microparticle comprises chemicallyfunctional groups that form covalent linkages with the antibiotic,antimicrobial or antiviral compounds, drugs or agents of the invention,preferably via a chemically or enzymatically cleavable linker moiety. Insuch embodiments, the cleavable linker moiety is specifically chemicallyor enzymatically cleaved within the particular infected phagocytic cellafter phagocytosis and is not degraded systemically or in uninfectedphagocytic cells. In preferred embodiments, the phagocytic cell isinfected with a tuberculosis-causing or other Mycobacterium-associateddisease-causing microorganism. In alternative embodiments, themicroparticle is conjugated with a multiplicity of antibiotic,antimicrobial or antiviral compounds, drugs or agents, or comprises amultiplicity of cleavable linked moieties, or both.

[0164] In a third embodiment of the invention, the microparticle isnonporous and the antibiotic, antimicrobial or antiviral compound, drugor agent is coated on the outside of the microparticle. Themicroparticle is further coated with a specifically-degradable coatingmaterial to control the specific release of the antibiotic,antimicrobial or antiviral compounds, drugs or agents in infectedphagocytic cells. In preferred embodiments, the phagocytic cell isinfected with a tuberculosis-causing or other Mycobacterium-associateddisease-causing microorganism. In such embodiments, a chemically orenzymatically-degradable coating covers the surface or outside extent ofthe microparticle, wherein the coating is specifically chemically orenzymatically degraded within the particular infected phagocytic cellafter phagocytosis and is not degraded systemically or in uninfectedphagocytic cells. In preferred embodiments, the phagocytic cell isinfected with a tuberculosis-causing or other Mycobacterium-associateddisease-causing microorganism. In preferred embodiments the antibiotic,antimicrobial or antiviral compound, drug or agent is an activated formof the compound, drug or agent, as defined herein. In alternativeembodiments, the microparticle is coated with a multiplicity ofantibiotic, antimicrobial or antiviral compounds, drugs or agents.

[0165] In alternative embodiments of this aspect of the invention, thecoating material is a non-specifically degraded coating that ischemically or enzymatically degraded within any phagocytic cell, whetheror not the cell is infected with a tuberculosis or otherMycobacterium-associated disease-causing microorganism. In theseembodiments, the antibiotic, antimicrobial or antiviral compound, drugor agent comprising said microparticles is provided as a “prodrug,”defined herein as an inactive or non-toxic form of an antibiotic,antimicrobial or antiviral compound, drug or agent, whereby the prodrugis converted or activated in a phagocytic cell by a protein having anenzymatic activity found specifically in phagocytic cells infected witha tuberculosis-causing or other Mycobacterium-associated disease-causingmicroorganism. In preferred embodiments, the prodrug is an activatedisoniazid derivative, such as INA, that is covalently modified at aposition involved in binding of the derivative to an NAD-requiringenzyme such as InhA. In these embodiments, the covalent modificationinterferes with and inhibits binding by at least 10-fold, so that theprodrug derivative is essentially inactive against NAD-requiring enzymeswithout activation. Activation of the prodrug is achieved specificallyin Mycobacterium-infected cells. In one aspect, such specific cleavageis due to a chemical linkage in the derivative that is labile within theinfected cell due to conditions caused by or that result from infectionof the cell with the mycobacteria. In another preferred aspect, suchspecific cleavage is due to an enzymatic activity which is producedeither by the mycobacteria itself or by the cell as the result ofinfection with said mycobacteria, wherein the linkage is enzymaticallycleaved by the enzymatic activity. In particularly preferredembodiments, the derivatizing group is a urea moiety that isspecifically cleaved in Mycobacteria-infected cells by amycobacteria-encoded urease. Alternatively, the prodrug of theantibiotic, antimicrobial or antiviral compound, drug or agent isspecifically activated by chemical cleavage under physiologicalconditions that are specific for phagocytic cells infected with atuberculosis-causing or other Mycobacterium-associated disease-causingmicroorganism.

[0166] In additional alternative embodiments, porous or non-porousmicroparticles are impregnated with or coated with a multiplicity ofantibiotic, antimicrobial or antiviral compounds, drugs or agents. Incertain embodiments of this aspect of the invention, the microparticlesare impregnated with or coated with a multiplicity of antibiotic,antimicrobial or antiviral compounds, drugs or agents, and then coatedwith a specifically-degradable coating material as described herein. Inother embodiments, the microparticles are impregnated with or coatedwith a multiplicity of antibiotic, antimicrobial or antiviral compounds,drugs or agents in prodrug (an inactive or non-toxic) form, and thencoated with a nonspecifically-degradable coating material andspecifically activated in phagocytic cells infected with atuberculosis-causing or other Mycobacterium-associated disease-causingmicroorganism. In yet other embodiments, the microparticles areimpregnated or coated with a first antibiotic, antimicrobial orantiviral compound, drug or agent, then coated with aspecifically-degradable or nonspecifically-degradable coating material,and then further coated sequentially and in an alternating fashion withan antibiotic, antimicrobial or antiviral compound, drug or agent thatcan be the same or different than the first antibiotic, antimicrobial orantiviral compound, drug or agent, and further coated with aspecifically-degradable or nonspecifically-degradable coating material,wherein is provided microparticles having a multiplicity of alternatinglayers of antibiotic, antimicrobial or antiviral compound, drug or agentand degradable -coating materials. The final coating is most preferablya specifically-degraded material that is degraded only in infectedphagocytic cells. In embodiments wherein any of the coating materialsare nonspecifically degraded, the antibiotic, antimicrobial or antiviralcompound, drug or agent uncoated thereby is provided in an inactive ornontoxic form that is specifically activated in a phagocytic cellsinfected with a tuberculosis-causing or other Mycobacterium-associateddisease-causing microorganism. These embodiments of the microparticlesof the invention permit the sequential, controlled, delayed or sustainedrelease of antibiotic, antimicrobial or antiviral compounds, drugs oragents or multiplicity thereof in a phagocytic cell infected with atuberculosis-causing or other Mycobacterium-associated disease-causingmicroorganism.

[0167] In all aspects of the invention, specific release or activationof the antibiotic, antimicrobial or antiviral compounds, drugs or agentsis dependent on specific chemical or enzymatic cleavage of the coatingor linker moieties inside cells infected with a tuberculosis-causing orother Mycobacterium-associated disease-causing microorganism afterphagocytosis of the microparticle, or specific activation of a prodrug(inactive) form of said compound, drug or agent. The specificity of thecleavage of the linker or coating moieties as provided by this inventionis the result of the combination of particular linker moieties orcoating materials which are selected to be specifically cleaved insidethe infected phagocytic cells. In one embodiment, such specific cleavageis due to a chemical linkage which is labile within the infectedphagocytic cell due to conditions caused by or that result frominfection of the phagocytic cell with the particular microbial pathogen.In another aspect, such specific cleavage is due to an enzymaticactivity that is produced either by the microbial pathogen itself or bythe phagocytic cell as the result of infection with said microbialpathogen, wherein the linkage is enzymatically cleaved by the enzymaticactivity.

[0168] Examples of such combinations resulting in specific release ofthe antibiotic, antimicrobial or antiviral compound, drug or agentcomponent of the compositions of matter and pharmaceutical compositionsof the invention within infected phagocytic cells include but are notlimited to a urea-based linker for use against a pathogen which producesurease (most preferably Mycobacteria spp.), as shown in FIG. 6.

[0169] In additional embodiments, the linker is a peptide comprising theamino acid sequence:-Ala-Xaa-CYS_(Acm)-Tyr-Cys-Arg-Ile-Pro-Ala-CYS_(Acm)-Ile-Ala-Gly-Asp-Arg-Arg-Tyr-Gly-Thr-Cys_(Acm)-Ile-Tyr-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys_(Acm)-Cys_(Acm)-(SEQ.I.D. No.: 1), wherein the microbial pathogen expresses an enzymaticactivity that specifically disables the endogenous antimicrobial peptidedefensin (most preferably Mycobacterium spp.); nicotinic acid amidescleaved by nicotinamidases; pyrazinamides cleaved by pyrazinamidase;allolactose linkages cleaved by β-galactosidase; and allantoate linkagescleaved by allantoicase (most preferably Mycobacterium spp.); a peptideof formula (amino acid)_(n), wherein n is an integer between 2 and 100,preferably wherein the peptide comprises a polymer of one or more aminoacids and the microbial pathogen produces a protease or peptidase, morepreferably wherein the peptide comprises a microbial-specific peptidaseor protease cleavage site; and hydrolases that specifically cleave sugarand other saccharide moieties. Most preferably, an activated isoniazidanalogue, such as INA, is derivatized with a urea moiety that isspecifically cleaved in Mycobacteria-infected cells by amycobacteria-encoded urease.

[0170] The antibiotic, antimicrobial or antiviral compounds, drugs oragents of this invention are useful in inhibiting, attenuating,arresting, combating and overcoming infection of phagocytic mammaliancells by pathogenic microorganisms in vivo and in vitro, particularlytuberculosis-causing species such as M. tuberculosis, M. africanum andM. bovis, as well as infection by M. leprae, M. avium, M.intracellulare, M. scrofulaceum, M. kansasii, M. xenopi, M marinum, M.ulcerans, M. fortuitumand M. chelonae. To this end, the inventionprovides methods for treating an animal having a disease or disordercaused by one of these microorganisms, wherein the antibiotic,antimicrobial or antiviral compounds, drugs or agents of this inventionare administered to an animal infected with a pathogenic microorganismthat acutely or chronically infects phagocytic mammalian cells. Inaddition, prophylactic embodiments and uses of the pharmaceuticalcompounds of the invention are provided, for inoculating vulnerablephagocytic cells prior to or roughly coincident with infection with apathological or disease-causing microorganism. The antibiotic,antimicrobial or antiviral compounds, drugs or agents of this inventionfor prophylactic or therapeutic uses are administered in a dosage andusing a protocol sufficient to have an antimicrobial effect in thephagocytic cells of the animal. In addition, pharmaceutical compositionsuseful in the methods of the invention are also provided, comprisingmicroparticles of the invention and a pharmaceutically-acceptablecarrier, adjuvant or excipient. Routes of administration include oral,ocular, buccal, intranasal, intravenous, intramuscular, parenteral,transdermal, and rectal. In particularly preferred embodiments, thepharmaceutical compositions of the invention are provided as an aerosolor other easily-volatilized form, for delivery for example to the lungas provided by conventional inhalers and other pulmonary drug deliverydevices and vehicles.

[0171] The following Examples illustrate certain aspects of theabove-described method and advantageous results. The following examplesare shown by way of illustration and not by way of limitation.

EXAMPLE 1 Development of Synthetic Procedures for Attaching ModelCompounds to Microparticles via Urease-Cleavable Bonds

[0172] In order to develop and assess efficient methods for conjugatingantibiotic, antimicrobial and antiviral compounds, drugs and agents tomicroparticles, model systems are constructed using fluorescent-dyecompounds as models for drugs. Using these model systems, procedures aredeveloped for attaching fluorescent dyes to both soluble compounds andmicroparticles to form the soluble dye-linked conjugates and dye-linkedmictoparticle conjugates, respectively.

[0173] A soluble dye-linked compound is synthesized comprising a dye anda cleavable carbamate group (shown in FIG. 3). The advantage of using asoluble dye-linked compound is that such conjugates can be rapidlysynthesized and characterized using conventional synthetic-organictechniques (e.g., ¹H and ¹³C NMR, mass spectroscopy, and HPLC analysis).After this soluble dye-linked compound is characterized, the rate of dyerelease via urease-catalyzed hydrolysis of the carbamate is measured.The soluble model dye-linked compounds are expected to act as substratesfor urease based on the substrate specificities of closely-relatedurease substrates reported by Sandaram and Laidler (1970, Canad. J.Biochem. 48: 1132) and Fishbein and Carbone (1965, J. Biol. Chem. 240:2407).

[0174] a. Synthesis of Soluble-Dye Linked Compounds

[0175] The soluble dye-linked compound synthesized herein is a mixedacetal of 3-phenyl propanal, as shown in FIG. 4. This compound isprepared by reacting 4-hydroxyl-7-nitro-2-1,3-benzoxadiazole (NBD, afluorescent dye) and cyanic acid with 3-phenyl propanal using thefollowing protocol. A nucleophilic addition of NBD by reaction of theNBD hydroxyl group with the aldehyde carbonyl of 3- phenyl propanal isperformed using conventional techniques (Finley et al., 1980, J. Org.Chem. 45: 694; Jencks, 1969, CATALYSIS IN CHEMISTRY AND ENZYMOLOGY,McGraw Hill: NY, pp. 490-496) to form the hemiacetal. Production of thehemiacetal is favored by using anhydrous conditions in an aproticsolvent such as tetrahydrofuran and in a two-molar excess of the dye.The hemiacetal is then converted to the mixed acetal by cyanic acidaccording to the procedure of March (1985, ADVANCED ORGANIC CHEMISTRY:REACTIONS, MECHANISMS AND STRUCTURES, 3^(rd) ed., J. Wiley & Sons, NY,pp. 791-792). Cyanic acid is formed by the dry distillation of cyanuricacid (Linard, 1938, Anorg. Allgem. Chem. 236: 200), and addition ofcyanic acid to the hemiacetal results in formation of the mixed acetalof NBD and carbamate. The mixed acetal is stable in aqueous and organicsolutions, and is purified by HPLC or by column chromatography.

[0176] b. Synthesis of Dye-Linked Microparticles

[0177] Dye-linked microparticles are prepared as follows. Biodegradablepolyacryl-starch microparticles with an average diameter of 1-2 mm(Borissova et al., 1995, J. Pharm. Sci. 84: 256-262; Stjärnkvist et al.,1991, J. Pharm. Sci. 80: 436-440) are used because they are notimmunogenic in mice (Artursson et al., 1986, J. Pharm. Sci. 75:697-701), and no immunogenic response was detected usingpolyacryl-starch microparticles linked by lysine to dinitrophenol(Stjärnkvist et al., 1991, ibid.). These and other literature resultsindicate that neither the microparticle nor the combination ofdrug-linked microparticle should elicit an immunogenic response.

[0178] Polyacryl-starch microparticles are derivatized with afluorescent dye (NBD) as follows. First, the starch molecules comprisingthe microparticle are derivatized with 4-aminobutyraldehyde diethylacetal and carbonyl diimidazole (CDI) to yield microparticle-urethanelinked butyraldehyde diethyl acetal (Stjärnkvist et al., 1991, ibid.).The derivatized microparticles are stirred in a solution of HCl inaqueous ethanol (pH 2) for 8 to 16 hr at room temperature, resulting inthe hydrolysis of diethyl acetal to the aldehyde. The resultingmicroparticles have an accessible aldehyde moiety available forconjugation with the fluorescent dye. This aldehyde-derivatizedmicroparticle is then further derivatized to form the mixed acetal ofNBD and the carbamate as described above for synthesis of the solublecompounds.

[0179] The number of NBD dye molecules bound to the microparticle can bedetermined by taking a known weight of the microparticle dye-linkedconjugate complex, hydrolyzing the carbamate bond with base,centrifuging the solution, and measuring the concentration of dye in thesupernatant by HPLC. Based on the average microparticle size anddensity, the average number of dye molecules bound to each microparticlecan be calculated.

[0180] c. Analytical Procedures

[0181] HPLC procedures for analysis and resolution of the fluorescentdye (NBD) and soluble dye-linked compounds are developed for analyzingcleaved fluorescent-dye compounds by urease enzymes.

[0182] In both cell-free and cell-dependent dye-release experiments, dyereleased from soluble compounds and from microparticle conjugates ismonitored by an increase in the absorbance and/or fluorescence of thesolution. In cell-free analyses, the concentration of dye released fromthe model compound and microparticle conjugates is measured aftercentrifugation of the insoluble material from the solublenon-particulate fluorescent dye, then measuring the concentration byHPLC. In cell-dependent cleavage experiments, cells are lysed with 0.1%Triton X-100 to release intact microparticles and cleaved fluorescentdye. The insoluble microparticle fraction is removed by centrifugation,and fluorescence of the cleared solution is measured to determine theconcentration of free dye.

EXAMPLE 2 Measuring Release of Fluorescent Dye From Soluble Dye-LinkedCompounds by Purified Bacillus Urease

[0183] Dye release from soluble conjugates and dye-microparticleconjugates is measured by incubating the dye-conjugates in the presenceof Bacillus pasterurii urease, obtained from Sigma Chemical Co. (St.Louis, Mo.). Urease activity is first assayed using standard procedures(Worthington Handbook, 1964, Worthington Biochemical Co., Freehold,N.J.). In this assay, urease activity is measured by coupling ammoniaproduction from urea hydrolysis to glutamate dehydrogenase (GDH). Adecrease in NADH concentration (measured spectrophotometrically) isproportional to the amount of glutamate formed from ammonia, which isproduced by hydrolysis of urea by urease.

[0184] This same assay procedure is used to determine if B. pasteruriiurease cleaves the carbamate linkage of the soluble dye-linkedcompounds. In this procedure, the mixed acetal produced as described inExample 1 is used as the urease substrate. Urease activity is measuredspectrophotometrically using the GDH/NADH assay described above. Ofparticular interest is the rate of hydrolysis of the carbamate byurease.

[0185] Carbamate linker cleavage of fluorescent dye from conjugatesmicroparticles is also assayed using B. pasterurii urease. In initialexperiments, cell-free studies are performed with the dye-conjugatedmicroparticles of the invention to determine time and concentrationprofiles (i.e., hydrolysis rates). In these experiments, known amountsof dye-linked microparticles are incubated with increasing amounts ofpurified urease at 37° C. to demonstrate the dependence of the cleavagereaction on enzyme concentration. In addition, the time course of thereaction is examined to confirm that cleavage products accumulate in atime-dependent fashion consistent with conventional enzyme-catalysiskinetics.

[0186] To determine if accumulation of hydrolyzed dye is the result ofurease activity, control experiments are conducted under identicalconditions and procedures except that urease enzyme is not added to thereaction mixture. In additional control experiments, the dye-conjugatedmicroparticles of the invention are incubated with a macrophage extractnot known to contain urease activity. In these experiments, a macrophagehomogenate from macrophages from uninfected mice is prepared in a 3:1dilution in 100mM Hepes buffer, pH 7.5. The macrophage homogenate isfirst assayed for urease activity using the above-described conventionalassay to confirm that the mouse macrophage homogenate does not containurease activity.

[0187] These experiments are also useful for comparing dye release ratesfrom the soluble dye conjugates and the microparticle dye conjugates.

EXAMPLE 3 Establishing Macrophage Cultures and Infecting Macrophageswith M. fortuitum and M. chelonei

[0188] In order to assay the capacity of infected macrophages tospecifically cleave carbamate linker-conjugated microparticles, in vitromacrophage cell cultures are developed and infected with thenon-pathogenic mycobacteria strains M. fortuitum and M. chelonei, andthen used to determine if the infected macrophages selectively releasedye from the dye-linked microparticle conjugates complexes.

[0189] a. Mouse Macrophage Cell Cultures

[0190] Cell cultures are established from either (i) bone-marrow derivedmacrophages from C57/BL6 mice (H-2b), or (ii) transformedmonocyte/macrophage isolated from C57/BL6 mice or BALB/c mice sources.10-12 week old female mice, purchased from Bantin-Kingman (Seattle,Wash.) are used for these experiments. Mice are housed in plasticmicro-isolater cages in a temperature- and humidity-controlledenvironment with a 12 hour light/dark cycle and fed Purina Lab Chow andwater ad libitum. Cages, bedding, and food are autoclaved prior to useand all cage changes and mice handling are performed in laminar air-flowhoods. All mice are quarantined for a minimum of one week beforeexperimental use.

[0191] b. Preparing the Cell Cultures Monolayers

[0192] Bone marrow derived macrophages (BMMF) from C57/BL6 mice (MHChaplotype H-2^(b)) and a monocyte/macrophage cell line (J774A.1; MHChaplotype H-2^(d), ATCC, Manassas, Va.) are used for these studies.These cell types permit investigation of the efficacy of the dye-linkedmicroparticles drug-delivery system in both primary cell cultureisolates as well as transformed cell lines. In addition, inbred C57/B16and BALB/c mice exhibit the Bcg^(s) phenotype that is more permissiverelative to infection with saprophytic and rapidly-growing mycobacteriasuch as M. chelonei, and M. fortuitum (Denis et al., 1990, J. Leuk.Biol. 47: 25-29; Radzioch et al., 1991, J. Leuk. Biol. 50: 263; vanFurth, 1990, Res. Microbiol. 141: 256; Nibbering et al., 1994, Scand. J.Immunol. 40: 187).

[0193] BMMF cell cultures are established by collecting bone marrowcells from the long bones from the hind limbs of donor mice andculturing these cells in 24-well tissue culture plates at 5-10×10⁵cells/mL/well in DMEM culture medium supplemented with 10% FCS, 30%supernatant from L929 cells (a source GM-CSF-1; L cells are cultured inDMEM with 5% FCS) and antibiotics (100 U/mL penicillin and 100 mg/mLstreptomycin sulfate). After 6-8 days at 37° C. and 6-7% CO₂,established BMMF monolayers are washed with DMEM and recultured in 10%FCS/DMEM without antibiotics for an additional 24 hours. Thereafter,BMMF monolayers are infected with viable M. fortuitum or M. chelonei asdescribed below.

[0194] J774.1 cell cultures are plated in 24-well tissue culture platesusing 2.5×10⁵ cells/mL/well in 5% FCS/DMEM with antibiotics (penicillinand streptomycin). After 18-20 hours at 37° C. and 6-7% CO, J774.1monolayers are washed three time with DMEM and recultured in fresh 5%FCS/DMEM without antibiotics, and immediately infected with viable M.fortuitum or M. chelonei as described below.

[0195] c. Infection of Cell Monolayers

[0196] BMMF and J774.1 cell monolayers are infected with viable Mfortuitum or M. chelonei. These mycobacterial species were chosen forthese studies because (1) they exhibit more rapid in vitro intracellulargrowth than other mycobacteria (Denis et al., 1990, ibid.; Radzioch etal., 1991, ibid.; van Furth, 1990, ibid.; Nibbering et al., 1994,ibid.), (2) they are opportunistic pathogens for mammals (Steven et al.,1992, Cornea 11: 500; Sing et al., 1992, Tubercle & Lung Dis. 73: 305)and therefore represent appropriate models for more pathogenicmycobacteria, and (3) they both produce urease (Wayne and Kuica, 1986,BERGEY's MANUAL OF SYSTEMIC BACTERIOLOGY, Williams & Wilkins) andtherefore present testable models for specific in vitro targeting of theinventive drug-delivery system.

[0197]M. fortuitum and M. chelonei are cultured in Middlebrook 7H9liquid broth to midlog phase (3-4 days) and aliquots frozen at −80° C.Frozen aliquots are thawed and CFU titers determined by plating serialdilutions (in sterile PBS with 0.1% Tween 80) onto Middlebrook 7H11plates. The optimal multiplicity of infection (MOI) for the cellmonolayers is determined in preliminary experiments, with mycobacteriaadded in 0.5 mL of 5-10% FCS/DMEM (without antibiotics). The optimal MOIis indicated by the maximal differential in mycobacterial CFU between 1and 48 hours following infection, as this differential enhances theability to detect urease activity.

[0198] Six hours following infection, cell monolayers are washed threetimes with warm DMEM (to remove extracellular bacteria) and reculturedat 37° C. and 6-7% CO₂ in 5-10% FCS/DMEM without antibiotics, or withgentamicin sulfate to inhibit growth of extracellular mycobacteria. Thisin vitro infection methodology has been successfully demonstrated inpreliminary experiments with slower growing mycobacteria. Although M.fortuitum and M. chelonei are rapidly growing mycobacterium, they stillgrow more slowly than most common bacteria. Therefore, a 24-28 hourperiod of infection for macrophage cell monolayers is required. Optimalin vitro infection of macrophages (of the Bcg^(S) phenotype) with M.fortuitum or M. chelonei permits a 6-fold increase in intracellularmycobacteria at 24-48 hours following infection. Therefore, pulsinginfected cell with microparticles at 12-18 hours after infection shouldprovide sufficient numbers of infected macrophages as well as sufficienttime for urease production by the intracellular mycobacteria, both ofwhich represent essential elements in evaluating this unique,microparticle-based drug-delivery system.

EXAMPLE 4 Measuring Urease-catalyzed Release of Fluorescent Dye inMycobacteria-infected Macrophage Cell Cultures

[0199] The functional competence of M. fortuitum- and M.chelonei-infected macrophages to selectively release fluorescent dyefrom a dye-conjugated microparticle is determines as follows.

[0200] A. Incubation of Infected Macrophage Cultures withFluorescent-Dye-Linked Microparticles

[0201] Mouse bone-marrow derived macrophages or J774 cells are infectedwith M. fortuitum or M. chelonei as described above. Purified infectedmacrophage cells are incubated with dye-linked microparticles at aconcentration of about 5-10μM. Microparticle uptake is determined bylysing a known number of macrophage cells and determining theaccumulated dye fluorescence in solution. The effect of microparticleuptake on functional competence of non-infected macrophages isdetermined by comparing the bactericidal capacity ofmicroparticle-pulsed and non-pulsed phagocytic cell populations againstsubsequent infection with the intracellular bacterial pathogen Listeriamonocytogenes (Peck, 1985, J. Immunol. Methods 82: 131-140; Drevets andCampbell, 1991, Infect. Immun. 59: 517-523; Drevets et al., 1992, J.Leuk. Biol. 52: 70-79; Barry et al., 1992, Infect. Immun. 60: 1625-1632)

[0202] B. Pulsing Infected Cells with Microparticles and Determinationof Enzyme Activity

[0203] At 12-18 hours following infection of cell monolayers, cells areincubated with a bolus of microparticles. The optimal size and number ofmicroparticles used for cell uptake is determined as described inExample 3. At 2 hours after pulsing with the microparticles, cellmonolayers are washed twice with warm DMEM and recultured at 37° C. and6-7% CO₂ in 5-10% FCS/DMEM without antibiotics. At 6, 12, and 24 hoursfollowing addition of the microparticles, monolayer cells are lysed,either hypotonically with sterile water or with detergent (2.5% saponinor 0.1% NP-40; the quenching effects of detergents on fluorescencedetection are evaluated prior to these studies). Cell supernatants fromthe lysed monolayers are clarified by centrifugation (10,000×g, 10 min)in microcentrifuge tubes equipped with 30 kilodalton molecular weightcut-off membranes. The relative fluorescence (as a measure of ureaseactivity) of the supernatants from these centrifugations is determinedby fluorescence spectrophotometry. Negative controls for theseexperiments consist of microparticle-pulsed, non-infected cellmonolayers and infected, but non-pulsed cell monolayers. Positivecontrols for urease activity are commercially obtained enzymepreparations.

EXAMPLE 5 Preparing Activated Isoniazid Analogs

[0204] An activated isoniazid analog is prepared as described by Quemardet al. (1996, J. Am. Chem. Soc. 118: 1561-2). ¹⁴C-Isoniazid is incubatedin the presence of H37R_(v), enoyl-ACP reductase and katG-encodedcatalase-peroxidase from wild type M. tuberculosis is incubated for 20hours in a solution comprising 2 μM MgCl₂, 6% glycerol, 10 μM NADH, 100μM isoniazid, 1.9 μM KatG and 9 μM NADH-specific enoyl-acyl carrierprotein (ACP) reductase. After incubation, the reaction mixture isapplied to a Pharmcia-PD-10 column, eluted and analyzed by liquidscintillation counting. Only in the presence of catalase-peroxidase aresignificant amounts of ¹⁴C-labeled isoniazid observed to co-elute withwild type enoyl-ACP reductase. The M. tuberculosis katG-encodedcatalase-peroxidase enzyme produces radicals in the presence ofisoniazid and hydrogen peroxide (Hillar & Loewen, 1995, Arch. Biochem.Biophys. 323: 438-446.) The fractions having radioactivity are combinedand dialyzed against water using a dialysis membrane having a 10,000daltons molecular weight cut-off. The aqueous solution containing¹⁴C-labeled isoniazid-NADH complex is lyophilized and the resultingwhite powder collected and characterized.

[0205] Alternatively, the method of Magliozzo et al. (1996, J. Am. Chem.Soc. 118: 11303-4) is used to produce an isoniazid-NADH analog. In thismethod, isoniazid (20 mM) is incubated for 3 hr in 0.015 M phosphatebuffer (pH 7.0) containing 10 mM NADH and 130 μM manganese (II) nitrate.The Mn⁺² cation has been reported to catalyze the aerobic decompositionof isoniazid in a radical-mediated mechanism (Ito et al., 1992,Biochemistry 31: 11606-11613). The isoniazid-NADH analog is isolated byHPLC chromatography using 50 mM ammonium acetate (pH 7) and a 0% -15%gradient of acetonitrile for elution. Fractions containingisoniazid-NADH analog are collected, concentrated and lyophilized toyield a powder.

EXAMPLE 6 Preparing Competitive and Irreversible Inhibitors ofLong-chain Enol Acyl Carrier Protein Reductase

[0206] Antimicrobial microparticles are produced comprising eithercompetitive, non-competitive inhibitors or irreversible, “suicidesubstrate”-type inhibitors of long-chain enol acyl carrier proteinreductase (InhA).

[0207] Competitive and irreversible inhibitors of ACP reductase aresynthesized according to the methods and protocols disclosed inco-pending U.S. Ser. No. 09/______, ______, filed July ______, 2000,Attorney Docket No. 99,296-A, incorporated by reference herein. Examplesof these compounds are shown in FIG. 5.

EXAMPLE 7 Preparing Isoniazid-NAD Analogue Prodrugs

[0208] Activated anti-mycobacterial compounds as disclosed in Example 6are modified to prodrugs that are specifically activated inMycobacterium-infected cells as follows, using INA as a specific exampleof the method. 100 mg (0.10 mmol) of INA is dissolved in 10 mL ofanhydrous pyridine, and 1.0 g (23.3 mmol) of cyanic acid (Linard, 1938;Merck Index, 1983) bubbled into the reaction mixture over 30 minutes,then stirred for 4 hr at 60° C. Excess pyridine is removed under highvacuum, then purified by HPLC, as described above. The resulting INAderivative has a urea functional group covalently linked to the 1-aminogroup of the adenine portion of the NAD component of INA (termed INAprodrug 1). This compound is characterized by ¹H NMR, ¹³C NMR, and fastatom bombardment/mass spectrometry (FAB MS).

[0209] Alternatively, 100 mg (0.10 mmol) of INA is dissolved into 10 mLof anhydrous pyridine and cooled to 0° C. under nitrogen. Trimethylsilylchloride (TMSCl; 5 g, 44.6 mmol) is added over 15 minutes, followed bystirring overnight at room temp. The excess TMSCl and pyridine areremoved under high vacuum. Anhydrous tetrahydrofuran (THF; 20 mL) isadded and the pyridine-HCI salt is removed by filtration. The THFfraction is cooled to −78° C., THF is added to keep the per TMS-INA insolution. N-Butyl lithium (1.0 mL of 0.10 M solution) is added dropwisefollowed by the addition of 1.0 g (23.3 mmol) of cyanic acid (Linard,1938; Merck Index, 1983) that is bubbled into the reaction mixture over30 minutes. The reaction is warmed and incubated at room temperatureovernight. The derivatized product is isolated by the addition of 1.0 mLof water in 5 mL of ethanol, stirring at room temp for 3 hr,concentrating the reaction mixture and purifying the product by HPLC asdescribed above. The resulting INA derivative has a urea functionalgroup covalently linked to the nicotinamide formamide group (termed INAprodrug 2). This compound is characterized by ¹H NMR, ¹³C NMR, and FABMS.

EXAMPLE 8 Analysis of Isoniazid-NAD Analogue Prodrugs

[0210] Prodrug forms of activated anti-mycobacterial compounds asdisclosed in Example 7 are tested to demonstrate Mycobacteria-specificactivation of said prodrugs, using the urea-derivatized INA compounds ofExample 3 as a specific example of the method.

[0211] Because the pyridine nucleotide binding site is highly conservedevolutionarily, it is expected that an inhibitor of a NAD-dependentbacterial enzyme is likely to inhibit mammalian NAD-dependent enzymes aswell. Commercially-available bacterial alcohol dehydrogenase (ADH) isassayed by the method of Zahlten (1980, Biochem. Pharmacol. 29: 1973-6)in the presence of the INA to determine a K_(i) for the compound. Tocontrol for unexpected effects on the 340 nm absorbance by the analogs,these results are verified using a colorimetric assay according to Fiblaand Gonzalez-Duarte (1993, J. Biochem. Biophys Methods 26: 87-93). Anunrelated enzyme, NADH-dependent glutamate dehydrogenase is assayedaccording to the method of Meredith and Schmidt (1991, Life Sci. Adv.Plant Physiol. 10: 67-71) to confirm the ADH-derived results on theinhibitory potential of these analogs. The experiments described abovewill be repeated with urea-modified NAD analogs. These compounds shouldnot have inhibitory activity, due to the alteration of portions of thecompound that interact with the NAD binding site of the enzymes.

[0212] Implicit in the proposed mechanism of anti-tubercular activity ofthese prodrugs is that the mammalian cell, most preferably phagocyticcells such as macrophages, remains functional and intact long enough forthe NAD analog prodrugs to kill the infecting mycobacterium. It istherefore essential to understand the degree of inhibition ofNADPH-dependent enzymes as well, since these are the mainly biosyntheticenzymes required for macrophage repair. The NADPH-dependent enzymesisocitrate dehydrogenase (Dedhia et al., 1979, Experimental Mycology 3:229-239) and malic enzyme (Mackall & Meredith, 1970, Anal. Biochem. 95:270-4) are used to examine the effects of the INA derivatives onNADP-utilizing enzymes.

[0213] The ability of bacterial urease to produce active enzymeinhibitors is tested by in vitro incubation of the urea-modified NADanalogs with urease, both commercially obtained purified enzyme andpartially purified bacterial urease obtained for these experiments.Urease release of inhibitors is evaluated in two ways. During incubationof the urea-modified analogs with urease under assay conditions, therelease of the unmodified NAD analog is monitored by HPLC (Anderson &Anderson, 1983, Anal. Biochem. 134: 50-5), a method used successfully toquantitate a number of NAD analogs. Secondly, the ability to generateenzyme inhibition in the test systems (ADH, GDH) after incubation istested and comprises the most compelling evidence that urease canactivate the urea-derivatized NAD analogues of the invention.

[0214] To demonstrate that urease cleaves the urea functional groups onINA prodrug 1 and INA prodrug 2 to yield INA, urease (ureaamidohydrolase [EC 3.5.1.5]) from M. tuberculosis(Clemens et al., 1995,J. Bacteriol. 177: 5644-52), M. fortuitum, M. chelonae, and Proteusvulgaris are tested as follows.

[0215] Urease activity is measured by coupling with ammonia production,from urea hydrolysis, to a glutamate dehydrogenase (GDH) reaction (asdescribed in the Worthington Handbook, 1994). The decrease in NADHconcentration (measured spectrophotometrically at 340 nm) isproportional to the amount of glutamate formed from ammonia, which isproduced by hydrolysis of urea. The concentration of the prodrug and INAare also monitored by HPLC.

[0216] The activated isonicotinic acid anion is recovered as a sodium orpotassium salt and used to impregnate a porous microparticle that isthen coated with a compound cleavable by a urease enzyme produced by aMycobacterium species. This embodiment of the invention is prepared asfollows.

[0217] Synthesis of Carbamate-Derivatized Polymer Comprising theMicroparticle Coating

[0218] A carbamate-derivatized polymer used to coat a microparticleimpregnated with a salt of an activated isonicotinic acid anionaccording to the invention is prepared as shown in FIG. 7.Commercially-available 2-deoxy-D-ribose (Sigma Chemical Co., St. Louis,Mo.) is converted to isopropylidene-protected Compound 2 using reactionconditions described by Renoll & Newman (1955,“D,L-Isopropylideneglycerol,” ORGANIC SYNTHESIS COLLECTIVE, Vol III,John Wiley & Sons, Inc.: NY, pp 502-4). A carbamate group is introducedusing a procedure described by Kurzer (1963, “Arylures I. CyanateMethod,” ORGANIC SYNTHESIS COLLECTIVE, VOl. IV, John Wiley & Sons, Inc.:NY , pp. 49-51. The isopropylidene protecting group is removed undermild acidic conditions by adjusting the pH to pH 1.0 with dilutehydrochloric acid to yield Compound 3. Sodium periodate oxidation (asdescribed by Schmid & Bryant, 1993, Org. Syn. 72: 6) of the vicinaldiols of Compound 3 in aqueous solution yields the acyclic dialdehydecarbamate intermediate that is reduced with sodium cyanoborohydrideunder mild conditions (between pH 3 and 4) to yield Compound 4. Compound4 is then converted to Compound 5 by the addition of 2.2 equivalents ofcommercially-available (Aldrich, Milwaukee, Wis.) allyl2,2,2-trichloroacetamidate and a catalytic amount of triflic acid atroom temperature in a mixture (1:1 ratio) of dichloromethane andcyclohexane (as described in Wessel et al., 1985, J. Chem. Soc. Perkin1: 2247-2250; Iversen & Bundle, 1981, J. Chem. Soc. Chem. Comm. 23:1240-1241). Compound 5 is isolated by column chromatography using a1″×16″ column containing 20 g of silica gel, Merck grade 9385, 230-400mesh, and eluted with a gradient of 1% ether in hexanes to 5% ether inhexanes, used iodine as the visual indicator thereof and is typicallyobtained in 13% overall yield and characterized by ¹H NMR and ¹³C NMR.

[0219] Polymerization of Carbamate-Derivatized Polymer

[0220] Compound 5 is polymerized in a two-step process developed byMarsella et al. (1997, Angew. Chem. Intl. Ed. Engl. 36: 1101-1103).Compound 5 is first converted to a macrocyclic intermediate bytemplate-directed ring-closing metathesis chemistry, then themacrocyclic intermediate is polymerized by ring-opening polymerization(ROMP) to produce a polymer of high molecular weight and viscosity (asdescribed in Ivin & Mol, 1997, OLEFIN METATHESIS AND METATHESISPOLYMERIZATION, Academic Press: San Diego). High molecular weightpolymer is obtained by performing the reaction in a concentratedsolution of monomer and the absence of metal ions (Marsella et al.,1997, ibid.). The polymer is characterized by ¹H NMR and ¹³C NMR andgel-permeation chromatography (GPC). GPC is used to determine thepolymer's average molecular weight (Mw), the number average molecularweight (Mn) and the polydispersity number (Mw/Mn).

[0221] The advantage of producing Compound 6 by ROMP instead of moreconventional polymerization methods, such as acyclic-diene metasynthesis(ADMET) is that the ROMP reaction proceeds by chain-growthpolymerization (so-called “living” polymerization), which gives highmolecular weight polymers even if the reaction does not go tocompletion. By comparison, ADMET polymerization is a condensation-typepolymerization that requires very high conversion percentages (>99%) ofthe starting materials in order to produce high molecular weightpolymers (as disclosed in Marsella et al., 1997, ibid.; Ivin & Mol,1997, ibid.).

[0222] Preparation of Microparticles for Polymer Coating

[0223] Biodegradable polyacryl-starch microparticles having an averagediameter of 1-2 microns are prepared as disclosed in Borissova et al.(1995, J. Pharm. Sci. 84: 256-262) and Stjarnvist et al. (1991, J.Pharm. Sci. 80: 436-440) and are used as the vehicle to deliver INHanalogs to infected macrophages. These microparticles are ideal forhuman use because the polyacryl-starch polymers are not immunogenic inhumans (Artursson et al., 1986 ,J. Pharm. Sci 75: 697-701). In amodification of the published microparticle preparation procedure, theINH analog (0.1% by weight) described in Example 8 is dissolved in thepolymer solution before precipitating the polyacryl-starch polymer into1-2 micron microparticles. A solution of polyacryl starch (1 wt % inacetone) containing 0.1 wt % of INH analog is forced through a 100micron aperture using 75 psi of backpressure. The polymer is sprayedinto a rapidly stirring solution of 25% water in ethanol. Themicroparticles formed thereby are filtered and dried under high vacuumfor 12 hr. The yield of the microparticles isolated in this way istypically 27% isolated yield.

[0224] Coating Microparticles with Urease-Cleavable Polymer

[0225] The carbamate-derivatized polymer prepared above is dissolved inacetone by continuous stirring for 30 minutes to give a 5 wt/v %solution. Triethyl citrate (20wt/wt % of polymer) is added as aplasticizer. Purified talc (25 wt/wt %) is added to the clear solution.Enteric coating of the INH analog-impregnated microparticles is carriedout using a Uni-Glatt® Air Technologies Inc. (Ramsey, NJ) fluid bedcoater. The coating polymer is sprayed by the bottom-spraying techniqueusing a Wurster column. The inlet and outlet air temperatures aremaintained at 30° C. and 40° C. respectively. The microparticles areisolated and used without further purification.

EXAMPLE 10

[0226] Antibiotic compounds of the invention are used as follows. Themicroparticles of the invention as described in the Examples above ornegative control (saline) are administered to an animal infected with atuberculosis-causing microbial pathogen using both optimal andsuboptimal dosages and the most appropriate route of administration.After an optimal time period (determined from the nature of theinfection), phagocytic cells are collected from the animal and testedfor infection with the tuberculosis-causing microbial pathogen.Phagocytic cells from peripheral blood or thoracic washings are isolatedusing conventional methods (Ficoll-Hypaque density gradientcentrifugation) and tested for the presence of infectioustuberculosis-causing microbial pathogens using conventionalimmunological, microbiological and biochemical testing protocols (seeLaboratory Test Handbook, Jacobs et al., eds., Lexi-Comp, Inc:Cleveland, Ohio, 1994; Clinical Laboratory Medicine, McClatchey, ed.,Williams & Wiklins: Baltimore, Md., 1994; Clinical Diagnosis andManagement by Laboratory, 18th Ed., J. B. Henry, ed., W. B. Saunders:Philadelphia, 1991).

[0227] It should be understood that the foregoing disclosure emphasizescertain specific embodiments of the invention and that all modificationsor alternatives equivalent thereto are within the spirit and scope ofthe invention as set forth in the appended claims.

What is claimed is:
 1. A composition of matter comprising an activatedantibiotic, antimicrobial or antiviral compound, a porous microparticle,and a coating material, wherein the porous microparticle is impregnatedwith the compound and said coated microparticle is further coated withthe coating material, and wherein the coating material is specificallydegraded inside a phagocytic mammalian cell infected with amicroorganism to allow release of the compound within the infected cell.2. The composition of matter of claim 1 wherein the microorganism is atuberculosis-causing microorganism and the compound is selected from thegroup consisting of isoniazid, activated isoniazid, rifampin,capreomycin, ethionamide, cycloserine, ciprofloxacin, amikacin,streptomycin, ethambutol, pyrazinamide, and inhibitors of long-chainenol acyl carrier protein reductase.
 3. A composition of matteraccording to claim 2 wherein the coating material is chemically degradedinside a mammalian phagocytic cell infected with a tuberculosis-causingmicroorganism.
 4. A composition of matter according to claim 2 whereinthe coating material is a substrate for a protein having an enzymaticactivity, said protein being specifically produced in a mammalian cellinfected with a tuberculosis-causing microorganism.
 5. The compositionof matter of claim 4 wherein the coating material is a substrate for aprotein produced by the infected mammalian cell.
 6. The composition ofmatter of claim 4 wherein the coating material is a substrate for aprotein produced by the tuberculosis-causing microorganism infecting theinfected mammalian cell.
 7. A method of killing a microorganisminfecting a mammalian cell, the method comprising contacting said cellwith the composition of claim
 1. 8. A method of killing atuberculosis-causing microorganism infecting a mammalian cell, themethod comprising contacting said cell with the composition of claim 2.9. A composition of matter comprising an activated antibiotic,antimicrobial or antiviral compound, a microparticle and a cleavablelinker moiety comprising two linker functional groups, wherein thecleavable linker moiety has a first end and a second end and wherein themicroparticle is attached to the first end of the linker moiety througha first linker functional group and the compound is attached to thesecond end of the linker moiety through a second linker functionalgroup, and wherein the cleavable linker moiety is specifically cleavedinside a phagocytic mammalian cell infected with a microorganism. 10.The composition of matter of claim 9 wherein the microorganism is atuberculosis-causing microorganism and the compound is selected from thegroup consisting of isoniazid, activated isoniazid, rifampin,capreomycin, ethionamide, cycloserine, ciprofloxacin, amikacin,streptomycin, ethambutol, pyrazinamide, and inhibitors of long-chainenol acyl carrier protein reductase.
 11. A composition of matteraccording to claim 10 wherein the cleavable linker moiety is chemicallydegraded inside a mammalian phagocytic cell infected with atuberculosis-causing microorganism.
 12. A composition of matteraccording to claim 10 wherein the cleavable linker moiety is a substratefor a protein having an enzymatic activity, said protein beingspecifically produced in a mammalian cell infected with atuberculosis-causing microorganism.
 13. The composition of matter ofclaim 12 wherein the cleavable linker moiety is a substrate for aprotein produced by the infected mammalian cell.
 14. The composition ofmatter of claim 12 wherein the cleavable linker moiety is a substratefor a protein produced by the tuberculosis-causing microorganisminfecting the infected mammalian cell.
 15. A method of killing amicroorganism infecting a mammalian cell, the method comprisingcontacting said cell with the composition of claim
 9. 16. A method ofkilling a tuberculosis-causing microorganism infecting a mammalian cell,the method comprising contacting said cell with the composition of claim10.
 17. A method for treating a microbial infection in a human whereinthe infecting microbe is present inside a phagocytic cell in the human,the method comprising administering a therapeutically effective amountof the composition of claim 1 to the human in a pharmaceuticallyacceptable carrier.
 18. A method for treating a microbial infection in ahuman wherein the infecting microbe is a tuberculosis-causingmicroorganism and is present inside a phagocytic cell in the human, themethod comprising administering a therapeutically effective amount ofthe composition of claim 2 to the human in a pharmaceutically acceptablecarrier.
 19. A pharmaceutical composition comprising the composition ofmatter of claim 1 in a pharmaceutically acceptable carrier.
 20. Apharmaceutical composition comprising the composition of matter of claim2 in a pharmaceutically acceptable carrier.
 21. A method for treating amicrobial infection in a human wherein the infecting microbe is presentinside a phagocytic cell in the human, the method comprisingadministering a therapeutically effective amount of the composition ofclaim 9 to the human in a pharmaceutically acceptable carrier.
 22. Amethod for treating a microbial infection in a human wherein theinfecting microbe is a tuberculosis-causing microorganism and is presentinside a phagocytic cell in the human, the method comprisingadministering a therapeutically effective amount of the composition ofclaim 10 to the human in a pharmaceutically acceptable carrier.
 23. Apharmaceutical composition comprising the composition of matter of claim9 in a pharmaceutically acceptable carrier.
 24. A pharmaceuticalcomposition comprising the composition of matter of claim 10 in apharmaceutically acceptable carrier.